BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - am Fachbereich II (Biologie/Chemie) der Universität Bremen Vorgelegt von Jan P. Eubeler Bremen im April 2010
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Biodegradation of Synthetic Polymers in the Aquatic Environment
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BIODEGRADATION OF SYNTHETIC POLYMERS IN
THE AQUATIC ENVIRONMENT
Dissertation zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften - Dr. rer. nat. - am Fachbereich II (Biologie/Chemie) der
Universität Bremen
Vorgelegt von Jan P. Eubeler
Bremen im April 2010
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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1. Gutachter Hr. Prof. Dr. Rudolf Amann (Universität Bremen)
2. Gutachter Fr. Dr. Sabine Zok (BASF SE Ludwigshafen)
1. Prüfer Hr. Prof. Dr. Michael W. Friedrich (Universität Bremen)
2. Prüfer Hr. Prof. Dr. Thomas P. Knepper (Hochschule Fresenius, Idstein)
Termin des öffentlichen Kolloquiums:
21. Juni 2010, 13:30 Uhr, Alter Hörsaal, MPI für Marine Mikrobiologie, Celsiusstr. 1, Bremen
Eidesstattliche Erklärung
gem. § 6 (5) Nr. 1-3 Promotions-Ordnung der Universität Bremen vom 14 März 2007
Hiermit erkläre ich, das ich
die Arbeit ohne unerlaubte fremde Hilfe angefertigt habe,
keine anderen als die von mir angegebenen Quellen und Hilfsmittel benutzt habe und
die den benutzten Werken wörtlich oder inhaltlich entnommenen Stellen als solche kenntlich gemacht
habe.
Großkarlbach, den 01. April 2010
Jan P. Eubeler
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
III|XVIII
"Bildung ist das, was übrig bleibt, wenn man alles vergessen hat, was man
gelernt hat. (…)"
(Werner Heisenberg)
„… und was man dann wiederum zu erlernen bereit ist.“
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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Acknowledgements
First of all, I would like to thank both my supervisor Dr. Sabine Zok (BASF SE) and Prof. Dr. Thomas P. Knepper
(Fresenius University of Applied Sciences) and my colleague and friend Dipl.-Ing. M. Bernhard for their help and
cooperation on this project and all related topics.
Furthermore I would like Prof. Dr. Allan D. Cembella (AWI Bremerhaven) for taking me as his doctoral student
and being my supervisor. I wish him all the best for recovering from his very unfortunate accident.
I also like to express my deepest gratitude to Prof. Dr. Rudolf Amann for taking over the position of Prof.
Cembella and supporting me through final phase of my dissertation. Thanks also to Prof. Dr. Michael W.
Friedrich for his expertise on my thesis. My gratitude goes also to Sara Kleindienst and Juliane Wippler for
being part of the committee.
I would like to express my gratitude especially to Helmut Schwarz, Stephan Hammer, Juergen Bachner, Karl-
Heinz Ullrich, Arnold “Lumpi” Gottwald, Helene Froehlich and Andy Haupt (GV/TC), for their support and help
every day in the Lab. Special thanks for the great time with you guys and the perfect Friday morning
“breakfast”. I will always remember that!
I am also most grateful to Dr. Christian Schütt and Hilke Döpke (BAH/AWI Helgoland/Bremerhaven) for the
cooperation, their intensive and unlimited help and the good weeks I spent at AWI BAH.
Special thanks go to Mrs. Frauke Ernst from University of Bremen. Thank you for your fast and constant support
in every administrative aspect.
I would like to thank BASF SE, especially GV/T as well as Mrs. Dr. Katrin Schwarz and her Team (E-EMV/Q) for
funding my project and for their support. It was a pleasure to work with you.
I also want to thank a lot of BASF staff for various help in my project:
Jens Kampioni for sending me all Pluriol E type and Pluriol E care samples, MSc Motonori Yamamoto for
supplying ECOFLEX, ECOVIO, MaterBi and PLA samples in various different versions. Thanks go as well to Dr.
Joachim Fischer, Dr. Inge Langbein, Dr. Klaus Taeger and Dr. Peter Reuschenbach for inspiring talks and ideas.
Thanks to Dr. Uwe Witt for supplying me with Oxo-PE samples and for discussions and literature and to Gert
Asselman from BTC Specialty Chemical Distribution N.V./S.A. Belgium for sending me samples of PEG &
MAPEG. Special thanks to Uwe Ruffer and Dr. Andrea Hörster from the Bioanalytical Department at BASF SE for
analyzing my marine samples. I also want to thank Prof. Dr. Andreas Künkel and his team for their input on my
review papers.
I want to express special gratitude also in the name of BASF to Mr. Wittner and his team at Luisenpark
Mannheim for their support and interest and for allowing myself to collect many, many seawater samples &
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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microorganisms from their aquarium! Also, I would like to thank those involved in sending seawater samples
from the North Sea near BASF Guesthouse at Sylt, Westerland to our laboratory for use in marine tests.
Thanks to Dipl.-Phil., Dipl.-Soz.-Päd. Sylvia Böttger from HS Fresenius, Idstein for her big support in my search
for literature. She has been always kind and never tired of sending me publications I could not get off the
Internet.
Thank you very much GDCh, SETAC, ASMS, DECHEMA e.V., ACS and all the other scientific societies. You were
all being very cooperative and supportive through all my time as an undergrad and grad student! This is
gratefully acknowledged and appreciated! Special Thanks to VCI/FCI for providing me with a scholarship for the
SETAG/GDCh PGS-Course in Ecotoxicology. The course is done very well and I would like to thank all involved
colleagues for their commitment.
All the people I have forgotten to mention here may please forgive me. You have also participated in this work
and you may remind me of that whenever you wish!
Last but not least I want to thank my mom and dad and also my grandparents for always listening and being
helpful and supportive. I am very grateful for many, many years of constant and unlimited support.
I would also like to thank Hendrik Jacob, Manuela Peschka, Matthias “Möcki” Möckl and Stefan Kappner, as
well as Holger Rinker, Frank Hess and Jürgen Diehl for the great hours we spent together (not only in the
“Promised Land”) having lots of fun! And thanks to the BOSS and all his colleagues and friends that have
delivered many days of beautiful reward being my winding wheel.
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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Abstract
Biodegradation of synthetic polymers can be a sophisticated property for intelligent and sustainable products
that offer complex benefits for specific applications. There are many entry paths for synthetic polymers that
can accumulate in the aqueous and especially marine environment and little is known about their
biodegradation especially in the aquatic environment. The difficulties with determining biodegradation in those
environments are based on the absence of appropriate methods and also the fact that these environments
often prove low biodegradation rates. It is also complicated to detect biodegradation on polymeric substances
because of the high molecular weight, water insolubility and difficult molecular structure making it hard to
detect biodegradation products.
This work provides an overview of the actual status of research regarding biodegradation, results and methods
describing biodegradation of biodegradable polymers. The main focus of this study is to find out if standard
biodegradation tests may be used for the evaluation of polymer biodegradation. Its aim is to identify difficulties
and problems with these tests and to compare the biodegradation potential and biodegradation pathways in
the marine and freshwater environment for a selection of polymer types.
It is also investigated whether molecular or structural properties of the polymers influence the biodegradation
in different environments and the possible pathways of biodegradation. Similarities and differences are
identified and on a selection of the biodegradation tests an investigation of the microorganism community is
performed to evaluate the use of molecular methods to discover influences of microorganisms on the
biodegradation of polymers.
Investigating available methods on how evaluation of biodegradation can be performed best in this special field
is a first step in a new direction. It may be important for further development of representative test
procedures. First facts on how biodegradation can be determined under the special circumstances are
established based on standardized methods which are adapted to the requirements
Three different types of carefully selected polymers and their degradation behaviour are evaluated based on
OECD test guidelines. Compounds were selected based water solubility/insolubility and biodegradation
potential:
∙ Poly(vinyl pyrrolidone) is selected because it is known to be recalcitrant and is investigated to observe
differences and similarities in different tests and media when no biodegradation can be observed
(“negative control”).
∙ Poly(ethylene glycol), a water soluble biodegradable polymer is investigated in broad molecular
weight distribution ranging from molecular weights of 200 to almost 60’000 g∙mol -1. Data from
different aquatic compartments are compared to establish differences systematically.
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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∙ The water insoluble biodegradable polyesters Ecoflex® and Ecovio® are investigated focusing mostly
on marine environment. In these cases the biodegradation potential as well as similarities and/or
differences between aqueous and compost environments (where they are known to be
biodegradable) are the main aspects of the study.
Both water soluble polymers are important mass production products that are used in manifold applications
and that also have higher potential to enter the aquatic environment from their applications and products
directly or indirectly.
The biodegradation of poly(ethylene glycol) especially shows the importance of systematic investigations and
the possibilities in application of the available test methods. It was shown that there are major differences
between freshwater (activated sludge, OECD 301) tests and those in marine (synthetic and native marine
water) tests. The differences range from the time of biodegradation of the same substances to differences in
the biodegradation graphs of reference substances and also to differences in the metabolic pathway as is
shown with sophisticated analytical techniques. The potential of biodegradation freshwater and marine tests is
shown for the first time systematically for the group of poly(ethylene glycols) ranging from 200 to almost
60’000 g∙mol-1
. This data shows differences in the applied methods and also the applicability of the marine
tests. As shown in the figure below there are significant differences of biodegradation in both test media (here:
activated sludge and sea water) and between the biodegradation curves based on the different types
measurement parameters (carbon dioxide evolution and dissolved organic carbon).
Biodegradation of poly(ethylene glycol) (4500 g⋅mol-1) in activated sludge and marine biodegradation tests
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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It is shown in this study that poly(ethylene glycol) biodegrades up to a specific molecular weight in freshwater
and marine environment after certain time to full extent (up to 60 kDa in freshwater and 15 kDa marine water).
Poly(ethylene glycol) biodegradation is investigated for the first time to this extend and biodegradation
pathways are postulated with the help of sophisticated analytical methods as shown in the figure below. It
seems interesting, that there are obviously two different pathways in marine water for poly(ethylene glycol) of
approximately <1600 g∙mol-1 and for poly(ethylene glycol) of approximately >1600 g∙mol-1
. It also seems
interesting that some microorganisms prefer lower substrate concentration and related biodegradation degree
is lower when increased substance concentrations are used. Different pathways for the biodegradation were
established depending on molecular weight distribution as shown in the following figure.
Pathways for poly(ethylene glycol) degradation in freshwater and seawater
The results obtained from the complex studies in this work show that the international guidelines can be in
some cases applied directly in other cases need to be adapted.
Neither the criteria from OECD 301 biodegradation tests nor those from OECD 306 tests are appropriate for
biodegradation tests of synthetic polymers. The test duration is in most cases too short especially for marine
biodegradation. Many substances that are degradable will fail to pass the test criteria even though they are not
recalcitrant. Longer tests but below 200-300 days provide more insight with better reproducibility. It is often
helpful when analytical tools are available to determine specifically the polymers tested and it is required that
more than one parameter is measured to gain more reliable data. However, it seems that marine tests take
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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much longer than other environmental biodegradation or simulation tests. This is disadvantage for these tests
in any application.
Also molecular or structural properties of the polymers do influence the biodegradation in different
environments as well as number of hetero atoms in the chain, specific behaviour of groups that hydrolize but
do not biodegrade etc. Possible pathways of biodegradation are confirmed and similarities and differences are
identified which supports some of the known statements in published literature on other biodegradation
research projects mostly in solid media.
In summary the following statements based on the biodegradation results especially with synthetic polymers,
can be made:
∙ Biodegradation in standard test systems and marine test systems can differ in kinetics and pathway of
biodegradation.
∙ The tests using CO2
∙ Because of the immense buffer capacity of marine sea water generally higher blank control values as
well as much more variation has been observed in the tests when compared with OECD 301 standard
tests.
free air in closed systems give stable conditions and variations can be kept low
during the first 200-300 days.
∙ The desired type of analytical procedure (DOC/DIC; BOD, CO2
∙ Marine tests show mainly far lower biodegradation when compared to freshwater/WW and soil or
compost.
) determines the length of the study and
needs to be considered. If possible more than one parameter should be measured at the same time.
∙ Marine medium can be prepared synthetically in the lab or native sea water can be used if treated
carefully and water samples should be stored at constant temperature.
∙ Tests using native water may have more impact and are closer to natural conditions but no significant
differences were observed compared to synthetic medium in this study.
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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Abstract (Deutsche Kurzfassung)
Der biologische Abbau von synthetischen Polymeren kann eine besondere Eigenschaft für intelligente und
nachhaltige Produkte darstellen. Es existieren mehrere Eintragspfade für Polymere in unserer Umwelt, eine
Anreicherung insbesondere im Meer kann vielerorts beobachtet werden und bisher ist über ihre biologische
Abbaubarkeit in der aquatischen Umwelt relativ wenig bekannt. Schwierigkeiten bei der Bestimmung des
biologischen Abbaus in den Umweltkompartimenten basieren auf dem Fehlen von geeigneten Methoden und
auf den besonderen Eigenschaften des Umweltmediums welches oftmals zu niedrigen Abbauraten führt.
Außerdem ist die Bestimmung biologischer Abbaubarkeit von Polymeren in Umweltmedien komplex, da diese
z.T. ein sehr großes Molekulargewicht und sehr komplexe Strukturen besitzen und außerdem oftmals
wasserunlöslich sind. Diese Arbeit gibt eine Übersicht über den aktuellen Stand der Forschung zum Bioabbau,
Ergebnisse und Methoden welche Bioabbautests und bioabbaubare Polymere beschreiben. Die Arbeit
konzentriert sich auf die Evaluation verschiedener Standardmethoden um festzustellen in welcher Weise die
Bestimmung von biologischer Abbaubarkeit von synthetischen Polymeren am geeignetsten durchgeführt
werden kann. Des Weiteren umfasst die Arbeit eine systematische Studie zur aquatischen Abbaubarkeit einiger
ausgewählter Verbindungen in Süß- und Salzwasser-Medien.
Es wird ebenfalls untersucht ob spezielle Parameter wie z.B. die molekularen oder strukturellen Eigenschaften
der Polymere die biologische Abbaubarkeit in unterschiedlichen Medien beeinflussen und ob Unterschiede
oder Ähnlichkeiten in den Abbauwegen auftreten. In einem ausgewählten Teil der durchgeführten biologischen
Abbautests wird zusätzlich mittels molekularbiologischer Screenings die Mikroorganismen untersucht um
Einflüsse derer und deren Gemeinschaften auf die biologischen Abbauprozesse zu identifizieren. Die
Untersuchung verfügbarer Methoden auf ihre bestmögliche Anwendbarkeit in biologische Abbautests unter
diesen speziellen Bedingungen ist ein erster Schritt in eine neue Richtung. Dieser ist sehr wichtig um in Zukunft
repräsentative Testmethoden zu entwickeln. Erste Erkenntnisse werden hier unter den besonderen
Bedingungen mittels standardisierten Methoden ermittelt welche nach Bedarf angepasst werden. Konkret
werden drei verschiedene Typen von Polymeren sorgfältig basierend auf deren Wasserlöslichkeit/-Unlöslichkeit
sowie deren Potential biologisch abbaubar zu sein, ausgesucht:
∙ Poly(vinyl pyrrolidone) dient als negativ Kontrolle da die Substanz wie aus der Literatur bereits
bekannt weitgehend persistent ist.
∙ Poly(ethylene glycol), ein wasserlösliches biologisch abbaubares Polymer wird einem breiten
Molekulargewichtsspektrum von 200 bis fast 60’000 g∙mol -1 untersucht. Die erhobenen Daten zu
Abbauraten aus verschiedenen aquatischen Medien werden so systematisch vergleichbar dargestellt.
∙ Die wasserunlöslichen biologisch abbaubaren Polyester Ecoflex® und Ecovio® werden hauptsächlich in
marinen Abbautests untersucht. Hauptsächliches Augenmerk wird auf Abbauraten, Ähnlichkeiten und
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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Unterschiede zwischen den aquatischen Medien sowie im Vergleich mit bekannten Daten aus Boden-
oder Kompostabbautests gelegt.
Die beiden wasserlöslichen Polymere sind bedeutende Massenproduktion die in vielfachen Gebieten
Anwendung finden und die beide direkt oder indirekt aus ihren Anwendungen heraus unbeabsichtigt in die
aquatische Umwelt gelangen können. Besonders die systematischen Untersuchungen zum Abbau von
poly(ethylene glycol) zeigen die Wichtigkeit solcher Experimente und die Möglichkeiten der Anwendung
bekannter Testmethoden. Es kann festgestellt werden, dass es signifikante Unterschiede im Abbau zwischen
Süßwasser (Kläranlagen Belebtschlamm, OECD 301) und Salzwasser (OECD 306) Tests gibt. Die Unterschiede
umfassen sowohl Differenzen in der Abbaurate, Geschwindigkeit und Abbaugrad im Vergleich zu
Referenzmaterialien bis zu Unterschiede im Abbauweg, wie mit geeigneten analytischen Methoden gezeigt
werden konnte. Das Potential geeigneter Süßwasser- und Meerwasserabbautests wird hier zum ersten Mal
systematisch vergleichend in einem für PEG weiten Molekulargewichtsbereich von 200 bis annähernd 60’000
g∙mol-1
ermittelt. Dies hat, wie in folgender Abbildung (Kläranlagenbelebtschlammtest vs. mariner Abbautest,
sowie Messparameter Kohlenstoffdioxidentwicklung vs. gelöste Kohlenstoffbestimmung) dargestellt,
Unterschiede der angewendeten Abbaumethoden sowie die speziellen Möglichkeiten in der Anwendung
mariner Tests zeigen können.
Biologische Abbaubarkeit von poly(ethylene glycol) (4500 g⋅mol-1
Es kann ebenso in der Untersuchung mit poly(ethylen glycol) gezeigt werden, dass die Polymertypen sowohl in
Süßwasser- (bis zu 60kDa) wie in Salzwassertests (bis zu 15kDa) nach einer bestimmten Zeit vollständig
) in Belebtschlamm- und marinen Abbautests
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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abgebaut werden In diesem Ausmaß wird der Abbau von poly(ethylene glycol) zum ersten Mal untersucht und
mögliche biologische Abbauwege werden, wie in der folgenden Abbildung dargestellt, ermittelt. Interessant ist
die Tatsache, dass zwei unterschiedliche Wege in marinem Medium beobachtet werden. Entsprechend jeweils
für solche bis zu einem Molekulargewicht von ca. <1600 g∙mol-1 und für jene über einem Molekulargewicht von
etwa >1600 g∙mol-1
. Es wird zusätzlich festgestellt, dass für manche Mikroorganismen einen niedrigere
Substratkonzentration begünstigend auf den biologischen Abbau wirken kann und dass die Abbauraten sinken
wenn die Substanzkonzentration im Test höher ist.
Abbauwege für poly(ethylene glycol) in Süßwasser- und Salzwasserabbautests
Besonders die systematische Untersuchung von poly(ethylene glycol) zeigt die Wichtigkeit einer solchen
Herangehensweise bezogen auf die Anwendbarkeit der Methoden. Wie die Ergebnisse zeigen, können die
Methoden der internationalen Richtlinien teilweise direkt oder mit leichten Anpassungen verwendet werden
um valide Ergebnisse zu erzielen. Im Gegensatz erzielt die Herangehensweise der Abbaustudien mit den
wasserunlöslichen Polyestern wesentlich mehr fehlerhafte und teilweise schwer einschätzbare Daten.
Weder die Kriterien aus OECD 301 Abbautests noch die aus OECD 306 Tests sind geeignet um biologische
Abbaubarkeit bei synthetischen Polymeren nachzuweisen. In der Regel sind die standardisierten 30 oder 60
Tage eine oftmals zu kurze Periode, welche sogar bei biologisch abbaubaren synthetischen Polymeren nicht
ausreichend sind um verwertbare Ergebnisse zu erzielen. Viele Substanzen welche ggf. biologisch Abbaubar
sind, würden hier insbesondere durch diese kurzen Tests durchfallen obwohl ein Abbau zu beobachten wäre.
Die Tests mit einer Laufzeit von 200-300 Tagen liefern oftmals deutlich bessere Ergebnisse bei guter
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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Reproduzierbarkeit. Es sollten wenn möglich immer mehrere Parameter bestimmt werden und zusätzlich
sollten analytische Methoden zur Verfügung stehen um polymere und eventuelle Abbauprodukte zusätzlich zu
den Standardparametern zu Bestimmen um sichere Ergebnisse zu erzielen. Trotzdem benötigen marine Tests
eine deutlich längere Zeitspanne in Vergleich mit anderen biologischen Abbautests oder Simulationstests,
welches eine deutliche Einschränkung für diese Tests in der Anwendung bedeutet Generell kann beobachtet
werden, dass spezifische Parameter wie z.B. molekulare oder strukturelle Eigenschaften, Molekulargewicht,
Anzahl an Heteroatomen oder Verzweigungsgrad der Kette der Polymere einen großen Einfluss auf die
biologische Abbaubarkeit in den unterschiedlichen Medien haben. Dies bestätigt weiterhin bekannte
Untersuchungen aus anderen Bereichen des biologischen Abbaus wie z.B. beim Abbau in Boden oder Kompost.
Die Erkenntnisse basierend auf den biologischen Abbautests im Besonderen mit synthetischen Polymeren
können wie folgt zusammengefasst werden:
∙ Biologischer Abbau in Standardtests und in marinen Tests kann sich deutlich sowohl in der Kinetik als
auch in den Abbauwegen (Metabolismus) unterscheiden.
∙ Tests welche mit CO2
∙ Wegen der immensen großen Pufferkapazität von Meerwasser beobachtet man meist höhere
Blindwerte und mehr Variabilität in den Messwerten als in vergleichbaren OECD 310 Standardtests.
freier Luft in geschlossenen Systemen durchgeführt werden liefern i.d.R. stabile
Bedingungen und geringere Variabilität innerhalb der ersten 200-300 Tage
∙ Die angestrebte analytische Methode (DOC/DIC; BOD, CO2
∙ Marine Abbautests zeigen oftmals weit niedrigere Abbauraten als vergleichsweise Süßwasser bzw.
Kläranlagentests oder solche in Boden oder Kompost.
) bestimmt die Länge und Dauer der
Studien ebenfalls und muss bei der Planung berücksichtigt werden. Wenn möglich sollten mehrere
Parameter parallel bestimmbar sein.
∙ Das marine Medium für die Abbautests kann sowohl synthetisch im Labor hergestellt werden also
auch aus der Umwelt als natives Medium entnommen werden sofern dieses sorgfältig behandelt und
bei konstanten Temperaturen gelagert wird.
∙ Tests welche mit nativem Meerwasser durchgeführt werden, haben vermutlich eine größere
Bedeutung weil diese näher an reellen Bedingungen sind. Allerdings werden im Rahmen dieser Studie
keine signifikanten Unterschiede zwischen den Tests mit verschiedenen synthetischen oder nativen
Medien beobachtet.
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
Vibrio sp. Curved rods. Bacteria common in coastal environments and associated with animal tissue
1 x 2 2.
Staphylothermus marinus
Cocci. Hyperthermophilic Archaea. 15 1800
Thiploca auracae Filamentous. Sulfur Bacteria 30 x 40 40’000
Beggiatoa sp. Filamentous. Sulfur Bacteria 50 x 160 1’000’000
Epulopiscium fishelsoni Rods. Bacteria symbiotic in fish gut. 80 x 600 3’000’000
Thiomargarita namibiensis
Cocci. Sulfur Bacteria 750 200’000’000
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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As the cell size increases, the volume (V) increases more rapidly than the surface area (SA) which shows that
the critical factor affecting nutrient uptake is the SA/V ratio. Prokaryotic cells with large SA/V ratios are more
efficient in nutrient uptake.
The explanation can be supported by studies using low nutrient media and increasing the nutrient concentra-
tion which did not lead to increase in sell size. But also if nutrients are severely limited as in most marine habi-
tats, selection will favour small cells because of their efficiency [27].
Cells use various strategies to increase their SA/V ratio. Especially spherical sells are the least efficient shapes
regarding diffusion of nutrients and therefore many marine microorganisms are long and filamentous shaped.
It is interesting that even though larger organisms are present in marine water, the smaller ones are the most
abundant ones (Table 3). That such small cells play a vital role in marine life has only been found out lately [27].
Unfortunately it is not clearly known whether small cell size is due to starvation or genotypically determined
and it is also unknown if cells size determines or influences the rate of biodegradation or whether biodegrada-
tion occurs or not.
Table 3 - Classification of plankton by size (additional information for bacteria: some filamentous Cyanobacteria and sulfur-oxidizing bacteria occur in larger size classes)
Size category Size range [µm] Microbial groups
Size
Femtoplankton 0.01 - 0.2 Viruses
Abu
ndan
ce
Picoplankton 0.2 - 2 Bacteria, Archaea, some flagellates
15 different polyester samples of P(3HB-co3HV) and P(3HB-co-4HB) were prepared and biodegradation was
investigated with a bacterium isolated from laboratory media and identified as Pseudomonas piketii. It was
found that the bacterium also grew on 3HB, glucose, fructose, citrate and succinate but only 3HB was able to
induce the PHA depolymerase enzyme apart from 3HB. The enzyme was purified and its molecular weight was
determined as about 40 kDa. The optimum activity was observed at pH 5.5 and 40°C. H-NMR analysis revealed
that the main degradation product of the P3HB polymers was 3-hydroxybutyric acid [232].
The biodegradability of P(HB-co-HV) blended with starch as well as mechanical properties were determined
under aerobic and anaerobic conditions via weight loss. The starch content ranged from 0 to 50% (w/w) in the
polymer tested. With increasing starch content biodegradation increases as well. A mixed microbial culture
degraded pure PHB in over 20 days but the 50% starch P(HB-co-HV) polymer was already gone in less than 8
days. The tensile strength declined from 18MPa to 8MPa while Young’s modulus increased from 1.525 MPa to
2.489 MPa, but the overall mechanical properties remained in a useful range. Also aerobic degradation was
faster than anaerobic processes [233].
In a study on P(HB-co-HV) with different portions of 3-HV reaching from 17 to 60 mol% it could be shown that
with increasing 3-HV content the water contact angle and hence hydrophobicity increases. It is widely accepted
that when increasing the side chain lengths of the constituents the surface hydrophobicity inclines as well.
Along with the higher 3-HV content it can be also observed by FTIR-ATR (attenuated total reflectance) and
CURRENT SITUATION ON BIODEGRADABLE POLYMERS IN THE ENVIRONMENT
55|191
calculation of the crystallinity index (CI) and amorphous index (AI), that AI values increase and CI values de-
crease along as the polymer becomes more and more amorphous. These findings suggest that the degradation
rate is more dependent on crystallinity than on hydrophilicity [234] seeming more important than the depen-
dence on molecular weight of PHB and P(HB-co-HV) degradation [235]. Similar findings were reported earlier
for PHB degradation already [236;237] and other structures [222]. It has been shown that a correlation of the
degree of crystallinity, morphology, glass temperature, mechanical properties and biodegradability exists for
PHA blends. PHB homopolymers are very brittle but when mixed with other biodegradable polymers mechani-
cal properties can be changed and relevant materials can be manufactured that can have similar properties as
PE or PP or others [238].
2.14.2 Polythioesters
Polythioesters are new class of biopolymers, which can be basically synthesized with the PHA biosynthesis
system. A study by KIM ET AL. [239] approached the issue of biodegradation of poly(3-mercaptopropionat)
(poly(3MP)) trying to isolate microorganisms being able to degrade the polymer. About 74 different environ-
mental samples were screened but neither bacteria nor fungi were found hydrolyzing poly(3MP). Also soil,
compost and activated sludge were applied to search for microorganisms probably non-cultivable and consid-
ering also microbial communities but again, even after an exposure of more than half a year, no poly(3MP)
degrading organisms were found [239].
2.14.3 Polyesters with synthetic aromatic and aliphatic components
Since this group of polyesters has shown biodegradable properties, the group has been investigated closely
through recent years [240]. Especially the degradation in soil and compost was the main feature of interest.
This is mostly due to the market situation for these materials since they have major applications in packaging
and agricultural industry. It was shown that there are already huge differences in biodegradation for soil and
compost compartments as shown in Figure 10 [96;212] for different aliphatic-aromatic polyesters.
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Figure 10 - Weight losses of aliphatic-aromatic copolyester films (100µm) in soil and mature compost; components: E = > 1,2-ethanediol. P = > 1,3-propanediol, B: 1,4-butanediol, A: adipic acid, T: terephthalic acid; numbers reflect the ratio of
aromatic/aliphatic acid component in mol%, (e.g., ETA38:62 copolyester from 1,2-ethanediol, adipic acid and terephthalic acid with 38 mol% terephthalic acid in the acid component)
It is not surprising considering the ecological differences of environmental compartments, that biodegradation
in aqueous media are much slower because of the different components and microorganisms [93;220;240-
242]. The architecture and biodegradation of different BTA Polymers, Bayer Tir 1874®, PHB and Bionolle® were
investigated in regards to biodegradation by microorganisms in compost and soil.
A commercially available lipase, Rhizomucor miehei has been chosen for the degradation in several test assays.
Also, a screening for polyester-degrading microorganisms has turned up, that especially Actinomyces sp. are
surprisingly effective in degrading aliphatic/aromatic copolyesters with up to 60 mol% of the aromatic compo-
nent [93].
The biodegradation of the aliphatic-aromatic polyester BIOMAX® was investigated in a lab-scale bioreactor at
58°C. The reactor was inoculated with microorganisms obtained from compost and supplemented with pow-
dered test substance as well as an additional energy source. After an acclimation period, the microorganisms
were capable of degrading the major components of BIOMAX® and degradation was monitored by laser diffrac-
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tion. The particle size distribution shifted to smaller sizes until the diameters were indistinguishable from bac-
teria. microorganisms types were investigated using 16S rRNA gene sequencing and the bacteria belonged to
about 35 different groups with the majority being new species [243].
The aliphatic-aromatic copolyester Ecoflex® has been reported to easily biodegrade under compost environ-
mental conditions [240;241]. The polymer was developed especially for applications using compost as the
route of disposal. Today these types of polymers offer very good combinations of biodegradation and material
properties and can be used for manifold applications. The tests were done under thermophilic conditions to
reproduce municipal or industrial composting facilities. The biodegradation was also described under moderate
environmental soil conditions with 29 strains of enzyme-producing soil bacteria, fungi and yeasts. A screening
procedure was developed and the results show that after 21 days of exposure the polymer could be degraded
especially by some of the microorganisms. Since the duration of these tests is rather short and moderate envi-
ronmental conditions pose more limited ability for biodegrading the polymer, Ecoflex® was observed to be
partially degraded within the timeframe. To study real environmental compartments the tests would require
lasting longer. The microorganisms preferentially degraded the bonds between aliphatic components and the
biodegradation, as expected, is faster for oligomers than for polymer chains. Degradation intermediates were
detected and identified by GC-MS as monomers of the co-polyester. GPC results suggest that exo-enzyme type
degradation occurs where microbes preferentially hydrolyze the ester bonds at the termini of polymeric chains
[244]. Using polymers based on aliphatic-aromatic constituents together with other polymers such as PLA or
PCL might lead to special co-polymers that offer increased biodegradability and good material properties de-
pending on the requirements of the application both for the consumer and the environment.
In contrast, biodegradation of Ecoflex in soil has been already investigated as well as in compost. Especially the
thermophilic environment promotes biodegradation. The study’s performed in soil take much more time, but
Ecoflex is mineralized as well only it takes at least over 150-200 days.
The group of biodegradable polyesters also contains multiblock poly(ether-ester)s based on PBS as “hard” and
poly(ethylene oxide) as “soft” and hydrophilic components. When the content of PEO (Mw≡1000 g⋅mol-1) is
varied between 10-50 w% material, structural, physical and biodegradation properties change. Biodegradation
was observed in phosphate buffer and a lipase from Candida rugosa. Weight loss of the samples was in the
range of 2-10 w% and significant molecular changes were confirmed by GPC to be up to 40% of initial values
leading to the conclusion that degradation occurs through bulk degradation in addition to surface erosion of
the PBS-PEO polymers [245].
The biodegradation of polymers containing lactic acid was observed to increase with the rising amount of lactic
acid when copolymers of lactic acid, terephthalic acid, and ethylene glycol were synthesized and biodegraded
by different fungal species (Aspergillus sp., Mucor sp., Alternaria sp. and Rhizopus sp.) [246].
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2.14.4 Mater-bi®, Eastar bio®
The biodegradation of PLA, PCL and others (such as PBS and PBSA) has been investigated mostly in compost or
soil environments [167;170;247].
, PLA and PCL
PCL is synthetic polyester that can easily be biodegraded by microorganisms. PCL-degraders are widespread in
the environment. PCL is degraded mainly by lipase and esterase [186]. PLA, also a biodegradable plastic is up-
taken by animals and humans. Its application in medicine is widely studied and extensively developed. The
general mechanism of degradation is thought to be non-enzymatic hydrolysis. The crystallinity seems to play an
important role in that process as well. Several enzymes can degrade the polymer as well such as proteinase K,
pronase and bromelain. Only few degrading microorganisms have been characterized yet. They are also not
thought of being very widespread in the environment [186].
Four polymers (MATER-BI®, EASTAR BIO
®, NATUREWORKS PLA®, PCL) were investigated in solid and liquid [248]
and soil media [224]. Cellulose and PE were used as positive and negative control. 20µm films and PCL powder
were tested. Quantitative tests were carried out following ISO 14851 and ISO 14853 in liquid phase. Qualitative
tests were carried out in solid phase (aerobic) and liquid phase (anaerobic) as described in ISO 14851, ISO
14853, ASTM G 21-90 and ASTM G 22-76. Quantitative assessment shows that MATER-BI (42%) was the most
degraded substance followed by PCL (40%), EASTMAN BIO (15%), and PLA (4%) after 28 days. Qualitative infor-
mation obtained shows that microorganisms largely colonized especially Eastman bio and PCL in solid phase
experiments. In liquid phase experiments no changes could be observed after incubation. But with pre and post
incubation characterizations the differences could be seen [248]. MATER-BI® biodegradation was also observed
for aerobic and anaerobic conditions using organic fractions of municipal solid wastes and anaerobic WWTP
sewage sludge. Within 72 days of composting in aerobic sludge the polymer was degraded to 27%. Anaerobic
degradation was faster and also the same in terms of biodegradation when compared to cellulose reference
within of 32 days [162]. These results indicate that MATER-BI® biodegradation is better in anaerobic than aero-
bic environment.
PLA biodegradation has been investigated mainly in compost and was found easily degradable to 80-100%
within 7 weeks similar to Avicell (Cellulose) [170]. When biodegradation was compared between real and simu-
lated composting conditions a more detailed understanding developed. Cumulative measurement respirome-
tric system (CMR) and gravimetric measurement respirometric systems (GMR) were compared and showed
similar trends for simulated composting. The results were around 75-85% after 58 days of exposure (DOE). In
real compost environment, biodegradation was correlated to molecular weight distribution shifts and break-
down. MW of 4100 g⋅mol-1 was reached after 30 days of exposure. Results match well with theory and biode-
gradation mechanisms but still with some variability [249].
Hydrolytic degradation of PLA/PEO/PLA triblock copolymers was investigated after polymerization of PLA in the
Presence of PEG 2000. The early stages of ester-bond cleavage occurred randomly along the PLA blocks. With
advancing degradation, a swollen hydro gel layer composed of PLA/PEO/PLA copolymers with short PLA blocks,
expanded from the surface. Once the polymer was placed in aqueous medium it absorbed large amounts of
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water. Short PLA blocks derived from degradation of parent long blocks as confirmed with NMR. The hydro gel
layer remained at the surface via hydrophobic interactions of micro domains. The degradation and material
properties of these polymers should depend on initial degree of polymerization, crystallinity, LA/EO ratio and
processing [250].
UV-Irradiation effects on enzymatic degradation of PLA were investigated by TSUJI ET AL. Therefore, amor-
phous and crystalline PLLA (PLLA-A and PLLA-C) films were investigated under UV-irradiation for 10 and 60h.
The Proteinase K-catalyzed enzymatic degradation was observed. Molecular weight of both PLA samples can be
altered when UV-irradiation time is changed. Weight loss of UV-irradiated PLLA films was similar or higher
when compared to non-irradiated samples. UV-irradiation is expected to cause chain cleavage and the forma-
tion of C=C double bonds. It seems that this effect accelerates the decrease molecular weight supports faster
enzymatic degradation [251].
Biotic and abiotic degradation of PLLA oligomers of molecular weight 260-2880 g∙mol-1 has been studied in an
aquatic aerobic headspace biodegradation test for six months. Water soluble dispensable PLLA’s (MW 260-
550 g∙mol-1) were biodegraded at temperatures of 25°C and also 58°C. The larger, crystalline and hydrophobic
oligomers (MW 550-2880 g∙mol-1) could only be biodegraded at 58°C. The average molecular weights de-
creased both during abiotic and biotic degradation. The surface and inner structures of biotic degradation PLLA
was more porous than those of abiotic experiments. These experiments show that abiotic hydrolysis is not the
only explanation, though essential, for PLLA degradation. Also enzymatic cleavage seems to play its role [252].
Different poly(L-lactide-block-ε-caprolactone-block-L-lactide) polymers were synthesized to investigate biode-
gradation and hydrolysis effects at pH 7.4 and 37°C. The rate of hydrolysis depends on a sensitive combination
of morphology and composition. The initial chain cleavage (day 0-7) was suppressed most by those systems
with the highest ε-caprolactone (CL) crystallinity. In addition, microorganisms secreting PCL depolymerase
(cutinase) show the ability to degrade systems with longer caprolactone sequence lengths. It appears that ini-
tial caprolactone crystallinity and overall composition controls the hydrolytic degradation since the PLLA phase
is more susceptible to random chain scission. It has been shown that wild-type Fusarium solani and Fusarium
moniliforme degraded those copolyesters with longer caprolactone sequence, while cutinase-negative strain
Fusarium solani (mutant strain without cutinase) does not [253].
Different lactic acid based polyesters were investigated under controlled composting conditions. Therefore,
poly(lactic acids), poly(ester-urethanes) and poly(ester-amides) were synthesized and the effects of different
structure units were observed. Ecotoxicological impact of the compost was evaluated. All polymers degraded
over 90% within 6 months. Toxicity was detected in poly(ester-urethanes) where chain linking of lactic acid had
been carried out with 1,6-hexamethylene diisocyanate. The other polymers, chain-linked with 1,4-butane-
diisocyanate, showed no toxic effects [254].
A method for rapidly testing if polymers are biodegradable using oxygen consumption as the observed parame-
ter and two consortia of fungi, one containing five and one three different fungi strains. Minor differences in
the consortium result in major differences in the ability of the consortium to utilize the polymer as carbon
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source. The exposure isolates biodegradation and therefore is excellent for reference material development for
biodegradable polymers. It is also possible to pre-treat samples to initiate other degradation mechanisms, such
as photo degradation, hydrolysis or physical deterioration. The test has the potential do indicate if a polymer is
or is not biodegradable, but a ranking with regard to biodegradability requires longer times of exposure than
this procedure allows. The method is quite sensitive, detecting small changes in organism’s metabolism. Re-
sults are at hand within mere days. A well defined consortium is needed, since small changes in composition of
the consortium results in markedly different results [255]. This test can be applied only if a sufficient under-
standing of microorganisms can be relied on since a change in consortium determines excessively the outcome
of the results. Therefore, the environment were the test is set up is crucial as are the used strains.
In addition PCL, PE and PCL/PE blends were investigated using a 5 microorganism consortium. Polymers were
exposed for 16 weeks in an NSM containing 0.8 mg∙mL -1 potato dextrose. After exposure the samples were
cleaned and analyzed for weight loss, changes in molecular weight (GPC), molecular changes (FT-IR) and tensile
strength. While tensile strength began to decrease in PCL samples after 1 week of exposure, the molecular
weight distribution showed no changes. FT-IR indicated a loss of amorphous PCL from the surface of the sam-
ples. PE samples were observed to be very recalcitrant and the consortium was not able to degrade the sam-
ples at all [256]. Systematic investigations on biodegradation of packaging materials made from PVA and PCL
are described in detail by HEINZ HASCHKE et al. [257-259].
Block copolymers (PCL-PEG and PCL-PEG-PCL) were synthesized using a PEG with 4600 g∙mol-1, and enzymatic
degradation was observed in a pH 7.0 phosphate buffer solution with Pseudomonas lipase. PEG introduction
increases Hydrophilicity in the molecule and also makes the molecule more amorphous, the crystalline parts
only being the PLA-blocks. The degradation of the homopolymer and also of the block-copolymers was ob-
served to be nearly the same. The assumption was made that this feature is due to the increased hydrophilicity
[260], but considering the study described for PHB degradation [234] it might also be possible that it is due to
crystallinity.
2.14.5 Cellulose and cellulose-based polymers, lignocelluloses, lignin
Recent studies of cellulose, lignocelluloses and lignin, all being major parts of plant biomass and inevitable to
carbon lifecycles, show that each polymer is degraded by a variety of microorganisms which produce a set of
different enzymes that work synergically [261]. Most of the cellulytic microorganisms belong to eubacteria and
fungi but some anaerobic protozoa and slime molds are able to degrade cellulose. The interactions lead to
complete mineralization under aerobic and anaerobic conditions. Hemicellulose is degraded to monomer su-
gars and acetic acid. The biodegradation needs additional accessory enzymes such as xylan esterase, ferulic and
p-coumaric esterase acting together to efficiently hydrolyze wood xylans and mannans. The biodegradation of
lignin is somewhat more complex since its high molecular weight and the complexity of its structure as well as
the insolubility delay biodegradation. Extracellular, oxidative and unspecific enzymes, releasing highly unstable
products being subject to many further oxidation steps, catalyze the initial steps of lignin depolymerization.
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This process has been referred to as “enzymatic combustion”. “White-rot”-fungi species are the most efficient
microorganisms known up to date, degrading lignin [262].
Lignocelluloses degradation was studied using 14C-labelled substrate in marine and freshwater tests and it was
observed that 14C was found partially in different sugars and also in mineralization product CO2. The total lignin
degradation was below 30% after over 600h [263]. Thermophilic anaerobic biodegradation of 14C-labeled Lig-
nin, 14C-labeled Cellulose and 14C-labeled Lignocellulose were studied for 60-days at 55°C. It was observed that
degradation enhances at these elevated temperatures and degradation rates were 10- 15-fold higher than in
previous studies at 25°C [264]. Later, 68 Basidiomycetes species were screened for enzymes involved in lignin
degradation. Laccase activity was found in 50% of the fungi, 40% expressed aryl-alcohol oxidase and 29%
showed availability of Mn-dependent polymerase. Laccase activity was highest obtained in the cultures and the
over two enzymes were active significantly lower [265].
2.14.6 Starch and starch-based polymers
The biodegradability of starch and inulin and respectively oxidized forms of dialdehyde derivates was studied in
biological oxygen demand and modified Sturm-tests. A higher degree of oxidation of dialdehyde starch and
dialdehyde inulin results in a lower rate of oxygen consumption and mineralization over the incubation period
(between 100% for starch and 25% for dialdehyde starch-100% oxidized; 60 day-Sturm-test and between 82%
for inulin and 20% for dialdehyde inulin-100% oxidized; 60 day-Sturm-test). It is also demonstrated that the
oxidized dialdehyde inulin derivates degrade far less than the equivalent starch counterparts do. The decrease
in degradation can be attributed to changes in the polymer structure due to intra- and intermolecular acetal
formation. Apparently, the oxidized starch and inulin derivates adopt different conformations, resulting in
different susceptibility to microbial attack [266]. Anaerobic degradation has also been studied, but not many
experiments have been performed up to now [267]. Few data on starch base plastics by white rot fungus P.
chrysosporium was reported [268].
2.14.7 Polyamides
OPPERMANN ET AL. Reported the degradation of bio-polyamides (poly(γ-glutamic acid)) by certain microorgan-
isms and in different media reaching from freshwater & soil to sewage sludge. Samples were analyzed with
viscosimetric means determining a decrease in viscosity over the time of incubation. The stability against pro-
teolytic attack was investigated using different protease enzymes. The ability of a non-adapted microbial com-
munity to degrade the test substance was tested in different media was investigated in enrichment cultures.
Twelve isolates were able to use poly(γ-glutamic acid) as carbon source. To verify degradation pathways, dif-
ferent polymers were used as carbon source. One isolate was investigated closer. During the first 20h of inocu-
lation only a small change in poly(γ-glutamic acid) concentration could be detected, but a significant decrease
in average molecular weight was observed. During the next 70h both, average molecular weight and concen-
tration dropped significantly. This observation of a two-phase degradation corresponds with an introductory
fragmentation and a subsequent degradation of the oligomers. The amount of free glutamic acid increased
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which could be determined with HPLC via the reaction with o-phthalaldehyde used in pre-column modification
for amino acid analysis [269].
2.14.8 Poly(aspartic acid)
The biodegradable, water soluble, synthetic polypeptide has gained attention for being used as dispersant,
detergent builder and in biomedical applications because of its biodegradation and environmentally friendly
properties. Already, some studies have reported the structure dependent degradation behavior in activated
sludge. Also a study regarding PAA degradation in river water and isolation of a degrading species has been
reported. Analysis of PAA was done with GPC and it can be determined that the isolated Sphingomonas sp.
strain degraded only low molecular weight PAA components but the cell extract could hydrolyze PAA polymers
up to 150’000 g∙mol-1 [270].
2.14.9 Poly(vinyl alcohol)
PVA is sort of a polymer with some special features regarding its structure and characteristics. It’s a vinyl poly-
mer with a main chain linked by only C-C bonds equal to those found in PE, PP, PS and also in specialty poly-
mers such as poly acrylic acid or poly(acryl amide). Among all vinyl polymers manufactured, PVA is the only one
known to be biodegradable by microorganisms [186]. PVA is water soluble but also has thermoplastic features.
It can be molded in various shapes such as containers and films. This feature is used to make water soluble,
biodegradable carriers for fertilizers, pesticides or herbicides and such. PVA degrading microorganisms are not
ubiquitous in the environment and almost all reported strains able to degrade PVA belong to the Pseudomonas
genus. Several enzyme systems have been reported to degrade PVA. The pathway was proposed to degrade
PVA first by the action of a dehydrogenase, to yield poly(vinyl ketone), which was subsequently cleaved by a
hydrolase enzyme to yield products with either methyl ketone or carboxylate termini (Figure 11). Several en-
zyme systems have been reported to degrade PVA. The carbon chain bond is always cleaved first of either a
dehydrogenase or an oxidase and than it is followed by hydrolase or Aldolase reactions [186].
Figure 11 - Biodegradation of poly(vinyl alcohol) by Pseudomonas sp.
Anaerobic degradation has also been studied, but not many experiments have been performed up to now
[267]. Mixed polymer films based on PVA, protein hydrolysate and glycerol were investigated in aqueous envi-
ronment using unadapted current mixed culture from WWTP’s. PVA was degraded in pure form only after 10
days of lag-phase. It was observed that when mixed polymers were used, the protein component and glycerol
were degraded first and PVA was degraded in the second stage. 1st order kinetics described the process and
when adapted organisms were used, lag-phase shortened and degradation occurred in one single step with a
1.5 fold increased breakdown rate. The number in PVA degraders during the process was observed to be 100
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fold increased when substrate was present. The addition of protein hydrolysate and glycerol enhanced biode-
gradation more than assumed from proportional regression of individual components [271].
2.15 Analytical methods for polymer determination
In environmental analysis many methods are established to day. They range from simple physical or wet-
chemical [272] tests to immunological tests and bioanalytical [273;274] arrays and further to sophisticated
methods [275] with complicated setups and procedures. Mostly three steps (sampling, separation or enrich-
ment and detection) are important and may be adapted to the task that is performed. Most methods described
are used for “small molecules”. Polymers are somewhat complex because of their often high molecular weight
and it is more complicated to analyze these compounds in environmental matrices.
Analytical methods used for evaluating biodegradation of polymers are based on the chemistry of biodegrada-
tion either in aerobic or anaerobic tests. The level of biodegradation may be assessed by accurately establish-
ing changes in concentration of the polymer (GPC, MALDI-TOF), the oxygen uptake (OxiTOP®), evolution of CO2
(conductivity, TIC/DIC), removal of carbon (DOC/TC) or the incorporation of the polymer into biomass (radio
labeling techniques) [89].
Substance specific analytical tools are powerful but often complicated. As a basis separations combined with
mass spectrometry provide a huge variety for evaluation of many different problems [276].
2.15.1 Comparison of degradation techniques and analytical methods
A wide range of analytical methods has been generally used for the analysis of different polymers. An overview
of several methods was presented [277;278], including sections about gas chromatography (GC), gel permea-
tion chromatography (GPC), high performance liquid and thin layer chromatography (HPLC and TLC), atomic
absorption and plasma emission spectroscopy (AAS and ICP), IR spectroscopy (IR), nuclear magnetic resonance
spectroscopy (NMR), surface analysis with scanning electron microscopy (SEM), reflection electron energy loss
spectroscopy and reflection high-energy electron diffraction (RHEED), X-ray photoelectron spectroscopy (XPS)
or electron spectroscopy for chemical analysis (ESCA) and further techniques. Also ultraviolet-visible spectros-
copy (UV-VIS), X-ray diffraction (XRD) and thermal analysis (TA) techniques were used and described. Mass
spectrometric methods for polymer analysis on synthetic are described as well [279-282] as is sample prepara-
tion and matrix/analyte effects [283]. Molar mass profiling and degree of polymerization/polydispersity deter-
mination can also be performed using solution capillary electrophoresis of DNA-polymer conjugates [284] for
uncharged water soluble polymers that can be uniquely conjugated to DNA. As shown, Table 7 provides an
overview of selected methods of biodegradation research with sophisticated analytical techniques.
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Table 7 - Methods for biodegradation research linked to analytical techniques
Method Polymer form (physiology, morphology)
Inoculum and degradation criteria monitored
Comments Selected references
Gravimetry Film or physical intact forms
Wide range of inocula from soil, water, sewage or pure
species from culture collections
Robust method, good for isolation of degrading microorganisms. High
reproducibility. Disintegration cannot be differentiated from
biodegradation
[105;246;249;285]
Respirometry Film, powder, liquid and virtually
all forms and shapes
O2 consumed or CO2 most adaptable to many materials. Specialized
instrumentation may be required. When fermentation is
the major mechanism of degradation, the method gives
underestimated results
produced under aerobic
conditions or CH4 produced under methanogenic
conditions
[105;138;140;163;249;286]
Surface hydrolysis
Films or sheets, pieces and others
Generally aerobic conditions, pure enzymes are used.
Hydrogen ions released are monitored as incubation
progress
Prior information about degradation of sample by
microorganisms and particular enzymes is needed for a target
specific test.
[105;201;222]
EIS Films or coatings resistant to water
Test polymers should adhere on conductive materials.
Electrochemical conductance is recorded
Sample must be initial water impermeable for signal
transduction. degradation can proceed quickly and as it is
registered no further degradation process can be
distinguished
[105;204]
Radio labeling All kinds of materials
Marine, soil, sewage, compost sediment etc.
Samples need to be 14C labeled [263;287;288]
GPC/SEC Virtually most polymers soluble in different solvents such as PEG, PVP,
Ecoflex, Ecovio
Freshwater, Saltwater, CO2 Problems with environmental samples because extraction
may be required
-balance, DOC
[289-291]
GC, GC/MS Ecoflex and others, PHB, Xanthan,
polysaccharide, Avicel®.
Requirement: small molecules. MWD
low!
Soil leachate, CO2 Molecular weight can be limiting factor for this type of
analysis!
-balance; compost
[125;241;292-295]
HPLC, LC/MS PEG, PVP, Requirement: small
molecules. MWD low
Freshwater, Saltwater, CO2 Molecular weight can be limiting factor for this type of
analysis
-balance, DOC
[289]
MALDI-TOF PEG, PVP, Ecoflex, Ecovio molecules
with higher molecular weight, synthetic polymers
Freshwater, Saltwater, CO2 Parameters optimized, important for polymer analysis
-balance, DOC
[125;289;290;294-298]
AFM Particles adhered or dispersed to a
substrate
Surface analytical procedure [79;235;299]
CURRENT SITUATION ON BIODEGRADABLE POLYMERS IN THE ENVIRONMENT
A large number of molecular methods have been developed for examination of microorganisms in complex
samples. Denaturing gradient gel electrophoresis (DGGE) is a widely used molecular fingerprinting method
[403-405] that separates polymerase chain reaction (PCR) generated DNA products. It was shown that DGGE
may not be suitable for detection of the most abundant organisms but more for numerical important organ-
isms in environmental samples [406]. The PCR of environmental DNA generates templates of differing DNA
sequence that represent many of the dominant microorganisms. PCR products from a given reaction are of
similar size (in terms of bp) and conventional separation by agarose gel electrophoresis gives only one single
DNA band however non-descriptive. DGGE removes this limitation by separating PCR products based on se-
quence differences that result in differential denaturing characteristics e.g. melting based on the GC content of
the DNA fragments in a gradient of DNA denaturants and an electronic field. The “melting domains”, defined as
stretches of base-pairs with identical melting temperature are sequence-specific [407]. The DGGE Procedure is
schematically shown in Figure 13.
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Figure 13 - The principle of denaturing gradient gel electrophoresis (DGGE). Double stranded DNA fragments from PCR are separated (PAA-gel with denaturing gradient). Increasing gradient of denaturants causes DNA to melt and separate while
moving through the gel. The GC-clamp attached to the 5’-end of the PCR fragment prevents complete denaturation
With the broad range of available PCR primers DGGE can be used to investigate broad phylogenies or target
organisms such as pathogens or xenobiotics degraders.
For initial fingerprinting analysis, the DGGE gel can be used directly. The bacterial profiles from the gel are also
useful when analyzing multiple samples over time, and to reveal profile differences. Time studies can also be
achieved when samples taken at different time points are compared on the same gel. To identify the origin of
DNA in gel bands of special interest, the bands can be recovered from the gel and sequenced. By sequencing
the band, the bacteria present in the sample can be determined, based on the DNA sequence information
(Figure 14).
Figure 14 - From sampling to bacterial detection and identification. DNA extractions, amplification and separation on a denaturing gradient gel before bands of interest is sequenced
The DGGE approach represents a rapid and reproducible method of studying population dynamics and to de-
termine cultivable and uncultivable microorganisms.
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2.18.4 Limitations of molecular methods
Generally, sampling and sample handling are known to produce biases. An obvious source of variability for
molecular methods is the extraction of bacterial DNA especially from a complex matrices or mixtures of cul-
tured bacteria. Environmental samples include proteins, humic acids, fulvic acids, enzymes and polysaccha-
rides, making it a difficult sample for all types of analyses. Interference, inhibition or enhancements of the
following analyses are often unknown.
PCR reaction itself provides many pitfalls. The main issue associated with the analysis of complex samples is the
presence of substances in the DNA mixture. These substances can inhibit or at least affect PCR amplification. To
minimize this effect, well established and verified PCR conditions and procedures were used in the present
study. Also, the band intensity may reflect the relative amount of particular bacteria or a bacterium for which
the PCR amplification is favored. Despite these and other limitations, DGGE is still considered as one of the few
techniques allowing a fast and reproducible microbial analysis of microorganism community.
2.18.5 Statistical support and database development
Today, it is very important that the information obtained with the described methods is available to the scien-
tific community. Databases are available and growing extensively today [408-410]. It has also been established
to support information using modeling techniques and combine all the information obtained and to evaluate
the data statistically [411].
2.18.6 Bacterial detection limits
One of the major problems and concerns for any quantitative bacteriological analysis is the detection limit. The
sensitivity of PCR-DGGE is based on the PCR reaction and its ability to amplify bacterial DNA. To get the best
possible results, the product must be as pure and as concentrated as possible. Theoretically, one cell in a 10µl
sample added to a PCR reaction of 100µl total volume, corresponding to 100 cfu∙ml-1, can be amplified by PCR.
Generally, the sensitivity in complex samples is reduced due to a wide range of inhibitory substances.
The fact that PCR does not distinguish between alive and dead cells is both an advantage and disadvantage.
PCR amplification is dependent on intact nucleic acid, rather than viable or non-viable cells. Positive PCR ampli-
fication and the presence of a PCR product do not imply that the target organisms were viable. PCR can detect
viable but non-cultivable (VBNC) and dead cells. This is a benefit in marine systems, since many microorganisms
seem uncultivable. In consequence PCR amplification may result in false positive results. Despite the possibility
of false positives, the predominant population will represent the cultivable bacteria during storage and the
bacterial profile of DGGE should be represented by bacterial DNA from the dominant, viable species rather
than the dead cells.
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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2.18.7 Polyester cleaving enzymes
Different BTA copolyesters have been investigated in an aerobic compost medium and especially Actinomyces
species and fungi could be determined as highly capable of cleaving the ester bonds. With these Isolates de-
gradation screening experiments can be accelerated considerably. Similar experiments with BTA polymers as
well as PHA samples were done under anaerobic conditions. About approx. 100 isolates could be obtained and
identified as mostly Clostridia [226]. Interestingly, the substrate spectrum of those microorganisms is rather
narrow. Those degrading natural polyesters cannot affect synthetic ones and vice versa. For PHA degradation
specific depolymerase enzymes are responsible which cannot degrade aliphatic polyesters, while less specific
lipase and hydrolase enzymes are responsible in degradation of the latter.
The isolation of a polyester-cleaving enzyme from a thermophilic Acetomyces, Thermomospora fusca (DSM
43793), which proved to be a rather efficient microbial isolate in cleaving polyester-bonds is described by
KLEEBERG et al. [182]. The cleaving enzyme is produced only after induction with insoluble BTA co-polyester. It
has a molecular weight of 25.5 kDa and a pI-value of 6.3. The enzyme showed a 10 times higher hydrolysis
activity compared to Pseudomonas sp. lipase and it was capable of cleaving aliphatic-aromatic polyesters as
well as pure aliphatic polyesters and polyester amides. The enzyme showed no activity regarding PHB, which
can be interpreted, that it seems to be a lipase. Further investigations show that BTA polymers were fully de-
graded to monomers after a short time. The cleaving organism did not further utilize the monomers.
A polyester-degrading extracellular hydrolase from thermophilic actinomycete Thermomospora fusca was pro-
duced and investigated. The excretion of the enzyme could be achieved with an optimized medium and only in
the presence of a polyester from 1,4-butanediol, terephthalic acid and adipic acid with around 40-50 mol%
terephthalic acid [180;412].
MATERIALS AND METHODS
75|191
3 Materials and methods
3.1 Chemicals and laboratory material
Utilized chemicals (salts, solvents, acids etc.) and material as well as molecular Markers, PCR equipment, En-
zymes and Kits were bought from Merck/VWR-International® Darmstadt, Germany or Sigma Aldrich, Germany
in analytical grade. Consumables and required materials are given in Table 8 and technical equipment is stated
in Table 9.
Table 8 - Consumables used in this work
Consumable supplies
Glass pasteur pipettes, open jet, length 150 mm, 230 mm (VWR®Glass fiber filter GF6, ∅ = 55 mm, 0.45 µm (Schleicher & Schüll,
Darmstadt, Germany)
®
Agar plates, sterile, VWR International, Germany
, Dassel, Germany)
Syringe filter: Spartan 13/0.45 RC, 0.45 µm, brown rim L (Schleicher & Schüll®
Eppendorf Tips, grey, yellow, blue, Eppendorf AG Hamburg, Germany
, Dassel, Germany)
Single-use fine dosage syringes Omnifix®-F 1mL (B.Braun®
G22-76 Resistance to bacteria Pseudomonas aeruginosa ATCC13388 Visual evaluation
D6691-01 Determination of aerobic biodegradation of plastics in
marine environment
Inoculum consists of a minimum of nine test organisms. Marine solution is
prepared in the lab following a standard procedure. Inoculum is verified by
standard identification test
CO2 OECD 306, ISO 16221
evolved
D6692-01 Determination of biodegradation of radiolabled polymeric plastics in seawater
Most ASTM methods use natural sea water as inoculum. No standardized
method is available. See also D7081-05
CO2 evolved, DPM counts (LSC), specific
radioactivity
D6340-98 Determination of aerobic biodegradation of radiolabled plastic materials in aqueous or
compost environments
Natural mixed culture or compost matrix CO2 evolved, DPM counts (LSC), specific
radioactivity
D6003-96 Determination of weight loss from plastic materials exposed to simulated
municipal solid-waste aerobic compost environment
Simulated compost, commercial compost seed
Weight loss
D7081-05 Standard specification for Non-floating biodegradable
plastics in the marine environment
-- -- --
3.4.3.2 OECD screening Tests
The OECD Guidelines [138] (Table 23) divide tests into three categories. The first consists tests for ready biode-
gradation. The second summarizes tests for inherent biodegradation and third comprises complex simulation
tests which are closest to native environmental compartments but rather expensive and complicated demand-
ing a lot of expertise. Most important for practical uses, are the tests determining ready biodegradability of
substances. These are the most stringent ones; offering only limited opportunities for biodegradation and ac-
climatization of the inoculum [140]. Nevertheless, simulation tests are becoming more and more important
today, since very complex risk assessment strategies require a broad knowledge on products and chemicals.
The ready biodegradability tests are based on the removal of organic compounds measured as dissolved organ-
ic carbon, catabolic CO2 and/or biological oxygen demand. Biological oxygen demand has the benefit of being a
direct biological parameter of aerobic biodegradation compared to dissolved organic carbon removal, which
indicates only the strict elimination of carbon from an organic source. The latter parameter therefore allows
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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only indirect conclusions to be made. Respirometric tests allow also poorly water soluble compounds to be
tested and also tests in automated systems, thus playing a role in modern biodegradability testing [140].
Table 23 - OECD standard test methods
OECD Code Purpose Involved microorganisms and key
features Parameters monitored
Similar to
TEST
S O
N R
EAD
Y BI
OD
EGR
AD
ABI
LITY
301A Static aerobic aquatic test using standard conditions.
(DOC-Die-Away-Test)
Standardized defined inorganic test medium and mixed microorganisms. Aerated and stirred test through 28 days and 3-4 samplings a week. Non
volatile and not significantly adsorbable test compounds. Water
solubility at 10-40 mg⋅L-1
DOC removal. Comparison of
DOC
DOC
(start) to DOC(end)
ISO 7827
.
EU 92/69/EWG
C.4A
301B CO2-Evolution Test. Static aerobic aquatic test using
standard conditions. Measurement of
biogenically evolved CO2 and comparison to ThCO
Standardized defined inorganic test medium and mixed microorganisms. Aerated and stirred test through 28 days and 3-4 samplings a week. For Polymers a modification with higher
buffer capacity, higher test temperatures and longer test
duration can be used.
2
CO2 ISO 9439 -evolution
EU 92/69/EWG
C.4C
301C MITI-I. Designed for use in Japan. With special
inoculum preparation and obligatory specific analysis.
Only for Japanese
region
EU 92/69/EWG
C.4F
301D Closed bottle test. Static aerobic aquatic test system using standard conditions.
Oxygen supply from test water. Oxygen measurement with
electrode. Low inoculum concentration. Comparison of BOD with ThBOD or COD. DOC removal
determination possible.
BOD measurement. In completely filled
bottles.
ISO 10707
EU 92/69/EWG
C.4E
301E Modified test. Static aerobic aquatic test using standard
conditions with low bacteria concentration (e.g. river
water)
Standardized defined inorganic test medium and mixed microorganisms. Aerated and stirred test through 28
days and 3-4 samplings a week
DOC removal. Comparison of
DOC(start) to DOC(end)
ISO 7827
.
EU 92/69/EWG
C.4B
301F Respirometric test. Static aerobic aquatic test using
standard conditions. Comparison of BOD to
ThBOD or COD
Test compounds which are water soluble or insoluble at the test
concentration Of 100 mg⋅L-1
BOD measurement. In closed
respirometers. Add. Information for water soluble
compounds by DOC removal
substance or ThOD.
ISO 9408
EU 92/69/EWG
C.4D
TEST
S O
N IN
HER
ENT
BIO
DEG
RA
DA
BILI
TY
302A Semi continuous activated sludge test (SCAS). Semi
static aerobic aquatic test system using organic test
compounds and easily biodegradable organic
medium.
Daily fill and draw of test vessel. Water soluble non volatile not
significantly adsorbable organic compounds at concentration
between 20 and 50 mg⋅L-1
DOC measurement before and after replacement to
determine ultimate biodegradation
within a test time of 26 weeks
DOC
ISO 9887
EU 88/302/EWG
C
MATERIALS AND METHODS
89|191
OECD Code Purpose Involved microorganisms and key
features Parameters monitored
Similar to
302B Static test (Zahn-Wellens-test). Static aerobic aquatic
test using standard conditions.
Higher concentration of TS and activated sludge (up to 1µg⋅L-1
DOC removal. Comparison of
DOC dry
substance). May be difficult to distinguish between abiotic
elimination by adsorption and biodegradation. For water soluble non volatile organic compounds between 50 and 400 mg⋅L-1 DOC
(start) to DOC(end)
ISO 9888
.
EU 88/302/EWG
C
302C MITI-II same as MITI-I (OECD 301C) but with
different test and inoculum concentration to improve
biodegradability
Only for Japanese
Government
SIM
ULA
TIO
N T
EST
303 Activated sludge simulation test. Continuously operated aerobic aquatic test system
using organic test compounds and easily biodegradable organic
medium.
Water soluble or satisfactorily dispersible non volatile organic compounds at concentration of
normally 10-20 mg⋅L-1
DOC or COD measurement in
influent and effluent of test and blank.
Test time of 12 weeks.
DOC
ISO 11733
EU 88/302/EWG
C
OTH
ER T
ESTS
304 Biodegradability in soil. Static test using soil as
medium and inoculum in a closed system. Radio
labeled TSs
Incubation time up to 64 days. Evolved 14 CO2 determination by alkali adsorption
and liquid scintillation counting.
306 Marine biodegradation. Static aerobic test using sea water. Shake flask (60 days) or closed bottle test with 28
day test duration.
Test compounds at concentration from 2-40 mg⋅L-1
DOC removal. Comparison of
DOC DOC
(start) to DOC(end)
.
307 Aerobic and Anaerobic Transformation in Soil
Systems
High Environmental relevance. Soil system from the environment
Substance specific analysis, radio
labeled analytes
308 Aerobic and Anaerobic Transformation in Aquatic
Sediment Systems
High Environmental relevance. Sediment/water system from the
environment
Substance specific analysis, radio
labeled analytes
309 Aerobic mineralization in SW - Simulation
Biodegradation Test
High Environmental relevance. SW system from the environment
Substance specific analysis, radio
labeled analytes
A study based on the results of a large number of chemical substances, compares the different biodegradation
tests (OECD 301, 303A, 302B) and the different measurement techniques to provide an assessment in terms of
passing or failing threshold criteria. The comparison revealed high consistency for CO2, biological oxygen de-
mand and dissolved organic carbon measurements. The compared data pairs formed a solid basis for a reliable
prediction of the carbon-based removal of chemical substances in WWTP’s [139].
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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3.4.3.3 ISO screening tests
With its increased use, the disposal of plastic wastes has become a major environmental issue. More and more
biodegradable plastics emerge as one of many available options to solve these issues. Since the working group
on biodegradability of plastics was created in 1993, rapid advances have been made in this area. The approach
and the development of standardized methods and definitions are described by SAWADA [417]. The following
table (Table 24) shows an overview of ISO standards [155] for biodegradation tests.
Table 24 - ISO standard test methods
ISO code Purpose Involved microorganisms and key
features Parameters monitored Similar to
TEST
S O
N R
EAD
Y BI
OD
EGR
AD
ABI
LITY
7827 Static aerobic aquatic test using standard conditions.
(DOC-Die-Away-Test)
Standardized defined inorganic test medium and mixed
microorganisms. Aerated and stirred test through 28 days and 3-4 samplings a week. Non volatile and not significantly adsorbable
test compounds. Water solubility at 10-40 mg⋅L-1
DOC removal. Comparison of
DOC
DOC
(start) to DOC(end)
OECD 301 A
.
9408 Respirometric test. Static aerobic aquatic test using
standard conditions. Comparison of BOD to
ThBOD or COD
Test compounds which are water soluble or insoluble at the test
concentration Of 100 mg⋅L-1
BOD measurement. In closed respirometer. Add. Information for
water soluble compounds by DOC
removal
substance or ThOD.
OECD 301 F
9439 CO2-Evolution Test. Static aerobic aquatic test using
standard conditions. Measurement of
biogenically evolved CO2 and comparison to ThCO
Standardized defined inorganic test medium and mixed
microorganisms. Aerated and stirred test through 28 days and 3-4 samplings a week. For Polymers a modification with higher buffer
capacity, higher test temperatures and longer test duration can be
used.
2
CO2 OECD 301 B -evolution
10707 Closed bottle test. Static aerobic aquatic test system using standard conditions.
Oxygen supply from test water. Oxygen measurement with
electrode. Low inoculum concentration. Comparison of BOD with ThBOD or COD. DOC
removal determination possible.
BOD measurement. In completely filled
bottles.
OECD 301 D
TEST
S O
N IN
HER
ENT
BIO
DEG
RAD
AB
ILIT
Y
9887 Semi continuous activated sludge test (SCAS). Semi
static aerobic aquatic test system using organic test
compounds and easily biodegradable organic
medium.
Daily fill and draw of test vessel. Water soluble non volatile not
significantly adsorbable organic compounds at concentration
between 20 and 50 mg⋅L-1
DOC measurement before and after replacement to
determine ultimate biodegradation within
a test time of 26 weeks
DOC
OECD 302 A
9888 Static test (Zahn-Wellens-test). Static aerobic aquatic
test using standard conditions.
Higher concentration of TS and activated sludge (up to 1µg⋅L-1
DOC removal. Comparison of
DOC dry
substance). May be difficult to distinguish between abiotic
elimination by adsorption and biodegradation. For water soluble non volatile organic compounds between 50 and 400 mg⋅L-1 DOC
(start) to DOC(end)
OECD 302 B
.
MATERIALS AND METHODS
91|191
ISO code Purpose Involved microorganisms and key
features Parameters monitored Similar to
SIM
ULA
TIO
N T
EST
11733 Activated sludge simulation test. Continuously operated aerobic aquatic test system
using organic test compounds and easily biodegradable organic
medium.
Water soluble or satisfactorily dispersible non volatile organic compounds at concentration of
normally 10-20 mg⋅L-1
DOC or COD measurement in
influent and effluent of test and blank. Test
time of 12 weeks. DOC
OECD 303 A
OTH
ER T
ESTS
10634 Guidance for poorly water soluble test compounds
Description of several techniques for preparation of poorly water soluble organic compounds and introduction to the test vessel
Dry mass [g∙L-1] (n=12)CFU per mL inoculum suspension (n=37) (right y-axis)
Figure 20 - Parameters of marine test medium for biodegradation tests prior to test phase
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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4.2 Microbiological analysis of marine media
In order to confirm that some microorganisms are present in the medium used for marine biodegradation tests
some microbiological investigations were performed (3.3.2). Generally one has to remember, that marine mi-
croorganisms are mostly famous for not being cultivable in the laboratory on agar plates or nutrient media.
Still, counting and estimating average colony forming units was used to confirm on short notice that media
used were comparable and for providing additional data. Generally the medium/inoculum samples were plated
on Difco Marine Agar plates undiluted and 1:10, 1:100 and 1:000. As a start, incubation temperature was inves-
tigated. Therefore samples were stored at different temperatures (6°C, 20°C and 30°C) during incubation time.
It could be observed that 20°C (RT) served best for the development of the cultures during the incubation time
(Table 37).
Table 37 - Effect of different incubation temperatures on colony forming unit counts
Inoculum 20°C Inoculum 30°C Inoculum 6°C
Mean CFU/mL 8.89∙10 8.08∙104 1.58∙104
Std. Deviation.
3
2.1∙10 7.1∙104 5.6∙104
Std. Deviation from the mean
2
1.2∙10 3.6∙104 2.8∙104
Std. Deviation from the mean [%]
2
13 44 18
The physico-chemical parameters and statistical evaluation of colony forming units’ counts are given in Figure
20. It can be seen, that cell counts vary quite a bit but over 50% of all values are in the range of 104. This allows
estimating how much inoculum suspension is used to spike the synthetic marine medium. Generally the results
have shown that a ratio of 1:10 (v/v, inoculum/synthetic medium) offers best results in biodegradation tests
performed in this study. A typical example for marine inoculum suspension after 7 day of incubation at 20°C on
agar plates is shown in Figure 21.
Figure 21 - Agar plate with marine medium after 7 days of incubation at 20°C (1:100 dilutions)
RESULTS
117|191
CFU counts were recorded each day for up to 7 days during aging of the medium for biodegradation tests. Ex-
periments with aged medium showed that no change could be observed when data was compared to non-
aged/fresh inoculum suspension.
Another experiment confirms the estimated correlation between growths of biomass/increase in colony form-
ing units during degradation of an easily degradable substance. PEG was used to investigate the effect and to
demonstrate that colony forming units can be used as a parameter to measure roughly the amount of cells in
marine medium although only about 1% of all marine microorganisms might be cultivable in the laboratory.
The experiment was done to a) verify correlation between colony forming units count and biodegradation and
b) to precondition marine microorganisms to PEG and to study if afterwards degradation would be much eas-
ier, specifically if degradation of PEGs with higher molecular weight distribution would occur more easily. The
last part will be discussed later in chapter 4.8. As can be seen in Figure 22 while TOC decreases because of
biodegradation of the PEG, colony forming units counts increases from 105 to almost 107.
duration [d]
0 5 10 15 20 25 30
CFU
∙mL-1
0
2e+6
4e+6
6e+6
8e+6
1e+7
0
20
40
60
80
100
Biod
egra
datio
n [%
]
CFU
TOC
Figure 22 - Correlation between number of colony forming units and degradation in biodegradation tests (PEG 2’000)
4.3 Molecular Biology Experiments with medium samples
A few samples of marine inoculum suspension taken from Luisenpark Mannheim, Germany and from Sylt
Westerland were investigated on molecular level as reference samples to biodegradation tests. The samples
are given in Table 31 and Table 32 (Samples 30-36). The samples were processed as described above and DNA
was sequenced. Figure 23 shows DGGE results for five samples taken from sea water aquarium at Luisenpark
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
118|191
and used for biodegradation tests. It is interesting that only few bands were observed and only few hits where
achieved from those samples.
Figure 23 - Denaturing gradient gel electrophoresis from Samples 30 to 34 with selected/identified bands (bands marked with arrows were submitted to sequencing and those with green frames were identified with forward and reverse sequence
in nBLAST)
The same samples and also 3 samples from Sylt Westerland were analyzed twice (Figure 24) to make sure that
results are reproducible. The results show again, that only few organisms could be detected. Along, some
bands were not accurately matched with existing data because only one of the two sequences matched results
or alignment was impossible because of limited data from sequencing.
Figure 24 - Denaturing gradient gel electrophoresis from Samples 30 to 36 with selected/identified bands (bands marked with arrows were submitted to sequencing and those with green frames were identified with forward and reverse sequence
in nBLAST. Bands that were only identified with low accuracy or where only one sequence (forward or reverse) matches database sequences are framed with red boxes)
RESULTS
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When the data is compared with results obtained from degradation experiments it is interesting, that the test
assays differ much from the analyzed media and there is also high variability between the test assays. Only a
few organisms were found in the medium samples and none of these organisms were detected in the test
assays at the end of the biodegradation experiments, but many other organisms were found.
During this research no method was found to accelerate marine biodegradation tests in any way. Higher con-
centrations of microorganisms and/or changes in the concentration of the test substance did not result in the
desired acceleration of the tests. The assumption can be made, that influences and interactions between dif-
ferent types of bacteria, fungi, viruses and others may also complicate the processes. If degraders would be
found, one would need to confirm degradation by isolating the organism and by the use of the cultures in
another experiment. If no degradation is observed there, or if a steady state is reached, this does not necessari-
ly mean that degradation is not possible. Also effects within the food chain or other population dynamics can
cause such results.
4.4 Basic carbon analysis statistics
To ensure correct carbon measurement (TC/TOC/TIC), the carbon analyzers used where cross checked and
calibrated on a random basis under GLP and ISO17025 certification. Before and after each analysis, standards
where run to confirm correct setup and working of the instrumentation. The data shown in Figure 25 gives a
good overview of the reliability and confirms the accuracy of the measurement. The data was collected during
the complete time of measurement of any biodegradation test.
TOC/TIC standard
TOC
2 (n
=39)
TOC
5 (n
=90)
TOC
20 (n
=337
)
TOC
40 (n
=330
)
TOC
100
(n=1
05)
TOC
400
(n=8
1)
TIC
4 (n
=585
)
TIC
20 (n
=426
)
TIC
80 (n
=467
)
TIC
100
(n=1
06)
TIC
200
(n=1
06)
TIC
400
(n=4
8)
conc
netr
atio
n m
g∙L-1
1
10
100
Figure 25 - Total carbon/total organic carbon/total inorganic carbon measurement and calibration statistics
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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4.5 Evaluation of blank control assays from biodegradation tests
To evaluate the results of biodegradation tests, it is imperative to estimate the influence of different factors to
the outcome of results. Therefore certain parameters were monitored and documented. To confirm each bio-
degradation test, blank controls and reference substances are submitted under the same conditions. All de-
termined/measured blank controls where cross checked to ensure the lowest possible variation between bio-
degradation tests. The data was obtained from basically the same test designs in each of the tests. Measure-
ments were made during different tests for each parameter throughout the duration of the test to show varia-
tion between many tests and also throughout the tests. It was distinguished between medium (marine and
WWTP) and measurement parameter/endpoint (DOC or CO2). Additionally, the NaOH solution blank control
(DIC) prior to each measurement interval when trapping flasks were switched and measured was statistically
evaluated. Figure 26 shows that generally dissolved organic carbon values of blank control assays are quite low
and very steady in both test media. Also NaOH solution shows only low variability. Since the data for NaOH
solution showed no significant difference between marine and WWTP tests, the data is given in this figure in
one box together for both media. It is interesting, that CO2 evolution from the blank control assays shows much
higher variation although carbon dioxide free air was used to aerate the test assays. This explains why carbon
dioxide evolution tests may give higher variation in the final results as well because blank control data is sub-
tracted in the calculation of the biodegradation degree of a tested substance. It is also interesting, that marine
biodegradation tests show higher variation in carbon dioxide blank control levels than in WWTP tests when
comparing 50% of the data (given by the box). However, the distribution of the data seems different. When
90/10 and 95/5 percentile is compared, WWTP tests show higher variation than marine tests.
DOC BC (marine, n 1172) CO2 BC (marine, n 1507) DOC BC (WWTP, n 836) CO2 BC (WWTP, n 992) NaOH BC (WWTP/marine, n 938)
mea
sure
d D
OC
or D
IC c
onte
nt in
bla
nk c
ontr
ols
[mg∙
L-1]
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
Figure 26 - Statistical evaluation of measured blank control data from marine and WWTP biodegradation tests (data based on tests of a maximum duration between 60 days of exposure for activated sludge tests and up to 180 days for marine
tests. Data of blank controls show variation characteristics that seem independent from experimental duration when single values are observed, which could be due to variations in a) airflow or b) quality of the carbon dioxide free air and c) varia-
tions caused additionally by the medium itself
RESULTS
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4.6 Reference substance statistics
In standard biodegradation tests aniline is mainly used as reference substance to proof that the medium has
basic biodegradation potential. A typical biodegradation curve for standard dissolved organic carbon-Die-Away
tests is given in Figure 27.
duration [days]
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Biod
egra
datio
n (D
OC
rem
oval
) [%
]
0
20
40
60
80
100
Figure 27 - Biodegradation of aniline in standardized OECD 301 A test (DOC Die-Away), n=42
The same biodegradation test but with measurement of carbon dioxide evolution (OECD 301B) gives the fol-
lowing results for reference substance (aniline) biodegradation (Figure 28).
duration [days]
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Bio
degr
adat
ion
(CO
2 evo
lutio
n) [%
]
0
20
40
60
80
100
Figure 28 - Biodegradation of aniline in standardized OECD 301 B test (CO2 evolution), n=100
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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If both figures are compared (see overlay in Figure 29Figure 30), it is obvious that the evolved carbon dioxide
can be measured only with a short delay of about a few days. This is due to the test setup and to the biodegra-
dation process. Also dissolved organic carbon based values are mainly higher at the end because formed bio-
mass or bound residues are not detected anymore.
duration [d]
-10 0 10 20 30 40 50 60 70
Biod
egra
datio
n/D
OC
rem
oval
[%]
-20
0
20
40
60
80
100
120
Figure 29 - Dissolved organic carbon removal and CO2
Therefore CO2 evolution is the more reliable parameter for biodegradation but it is also the more complicated
one to determine and is much more prone to interference as can be seen in the following paragraphs. In ma-
rine biodegradation tests aniline can be used as well but mostly Na-benzoate is taken instead. To confirm dif-
ferences between both substances within the same test medium, especially in marine tests, a biodegradation
test in marine medium (OECD 306) was performed with the two substances over 28 days as well as a standard
WWTP-test. The results of the marine test are shown in
evolution curve of aniline biodegradation in WWTP biodegradation tests (overlay)
Figure 30.
It is interesting, that in marine tests, aniline degradation takes longer than Na-benzoate biodegradation, while
in activated sludge tests biodegradation of both substances occurs within the same timeframe. The biodegra-
dation kinetic for benzoate in marine tests is almost comparable to kinetic of aniline in WWTP standard tests. If
Na-benzoate and aniline are submitted to a biodegradation test using WWTP activated sludge as inoculum
(OECD 301), aniline and Na-benzoate show biodegradation rates that differ much less than in marine tests. The
rate and biodegradation degree in WWTP test is similar for both test substances. No differences can be de-
RESULTS
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tected with the used measurement techniques. Biodegradation of those substances in WWTP tests occurs very
rapid in WWTP activated sludge which is why no difference can be observed.
26’700 -- 412 no biodegradation 26642 1.4E-06 40 no biodegradation 605
57’800 -- 412 no biodegradation 56695 6.5E-07 40 no biodegradation 1288
Further on, Figure 35 shows the results (n = 2) of the combined CO2/DOC Test obtained from degradation stud-
ies in freshwater with WWTP sludge inoculum. All PEGs with a molecular weight of up to 57,800 g∙mol-1 are
biodegradable in the given conditions. The PEGs can be divided into two groups: one group, the PEGs with
molecular weight from 250 to 14’600 g∙mol-1, was fully biodegraded within 20 d. They had a lag-phase of up to
5 d; their graphs in the combined CO2/DOC Test are similar in evolution. No differences concerning the biodeg-
radation could be seen for these PEGs. The second group includes the PEGs 26’600 and 57’800, which had a
longer lag-phase of approx. 22 d and were completely degraded within 45 d and 65 d, respectively. Evidently,
the biodegradation of PEGs having an molecular weight > 14’600 g∙mol-1required much more time than that of
PEGs with shorter chains but even PEG 57’800 was entirely biodegradable.
For PEGs up to molecular weight 57’800 g∙mol-1 in freshwater media, the time taken for PEG degradation to
reach the plateau phase generally increases with increasing molecular weight because the biodegradation rate
(per time) decreases. The biodegradability (the part of the test substance, which is completely mineralized and
characterized by the height of the plateau) is not affected by this. This is a new result because it is generally
accepted that the biodegradation degree in freshwater decreases with increasing molecular weight [188;200].
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Figure 35 - Aerobic biodegradation (expressed as DOC removal in %) of PEGs in freshwater using WWTP sludge inoculum, 0 - 65 d (n = 2). Results were obtained from the combined CO2
Aerobic biodegradation of PEGs in artificial seawater using marine bacteria is possible (
/DOC Test
Figure 36) but there are
differences compared to freshwater media. The graphs of PEG 250, 970 and 2’000 have the same trend with a
lag-phase of not more than 6 d, which is similar to freshwater media. These short-chain PEGs were fully biode-
graded within 37 d. The PEGs with longer chains, PEG 4’500, 7’400, 10’300 and 14’600, all had a lag-phase of
around 20 d until the biodegradation started. Their graphs are quite similar initially but then they vary, leading
to different results for biodegradation. PEG 4’500 is completely degraded after approx. 100 d whereas PEG
7’400 needs around 130 d. PEG 10’300 has reached only 80 % dissolved organic carbon removal after 180 d and
the degradation of PEG 14’600 has terminated after 50 d when dissolved organic carbon removal exceeded
40 %. Thus PEG 10’300 and 14’600 are not completely degraded in seawater media after 180 d, which is in
complete contrast to the freshwater media in which they were entirely biodegraded within 20 d. PEG 26’600
and 57’800 were not degraded in seawater for a period of 135 d. Neither significant dissolved organic carbon
removal nor CO2 production was observed (Figure 36). This is contrary to freshwater media in which a full bio-
degradation of these PEGs could be seen. The reference compound sodium benzoate in the reference test
systems of PEG 26’600 and 57’800 was degraded within 16 d indicating an active microbial population at the
beginning of the test series.
As a result of PEG degradation in seawater media, the time required for it increases whereas the biodegradabil-
ity decreases with increasing molecular weight. This was found for freshwater media [188;200] and can be
transferred to seawater media.
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Figure 36 - Aerobic biodegradation (expressed as DOC removal in %) of PEGs in artificial seawater using marine inoculum, 0 - 180 d (n = 2). Results were obtained from the combined CO2
The results based on evolved CO2 from CO2 evolution test (data not shown) and combined CO2/DOC Test was
similar for both test systems. The evolved CO2 production did not reach 100 % of calculated theoretical CO2
production, but was in a range between 70 and 95 %. The lag-times of graphs based on CO2 Evolution were
always longer than those of graphs created by dissolved organic carbon removal with differences being in the
region of 1 to 3 d.
/DOC Test
Figure 37 is given as an example for these findings and this “discrepancy” should be consid-
ered in tests of water-insoluble polymers when no dissolved organic carbon can be measured.
Figure 37 - Comparison of Measurement Parameters DOC and CO2 in PEG 4500 g⋅mol-1 biodegradation tests using marine and activated sludge inoculum
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The biodegradation tests are based on the determination of sum parameters. With the results obtained, it is
possible to draw conclusions about the differences in biodegradation of PEGs in freshwater and seawater me-
dia in terms of time and chain length. It is impossible to compare the degradation pathways in both media of
PEGs having the same molecular weight distribution, which may help to understand the difference between
the biodegradation processes in the two media [289].
The results obtained from the biodegradation tests were confirmed using sophisticated analytical techniques
[289]. In summary, the fate of each homologue of polydisperse PEG 250 and PEG 970 for freshwater and sea-
water media is shown in Figure 38 and Figure 39.
a)
b)
Figure 38 - Fate of individual homologues during aerobic biodegradation (0 - 23 d) of polydispersed PEG 250, measured by (+)-ESI-LC-MS. (a) freshwater media, (b) Artificial seawater media.
The peak intensity of each PEG homologue, measured as PEG-Na adduct by (+)-ESI-LC-MS, was set in relation to
the peak intensity of the PEG homologue with the highest intensity. This illustration is limited due the different
detector responses for the homologues of PEGs. Depending on the time, the change in intensities of each PEG
homologue can be observed. At the beginning of the dissolved organic carbon removal test, the intensities of
all homologues show the typical pattern of a polydispersed mixture. As can be seen for both media, the degra-
dation of PEG 250 (Figure 38) and PEG 970 (Figure 39) started simultaneously with all PEG chains leading to a
shift in the molecular weight distribution towards the short-chain homologues. The long-chain PEGs are com-
pletely degraded by gradual splitting of C2-units off the chain. This is a common accepted pathway of aerobic
PEG degradation in freshwater media [84;192], which now can be transferred to seawater media for PEG 250
and PEG 970. During degradation, short-chain PEG homologues are generated from the long-chain PEGs (Figure
38 and Figure 39). The amount of short-chain PEG increases in both media and decreases again when degrada-
tion proceeds (Figure 39). The aerobic biodegradation of polydispersed PEG 300 in freshwater with formation
of short homologues is known [196]. However we found out not only the biodegradation of PEG 250 occurs in
the same way for freshwater and seawater media but also PEG 970.
01
36
912
1416
2023
n = 3n = 4
n = 5n = 6
n = 7n = 8
n = 9
0102030405060708090100
Rel
. pea
k in
tens
ity [%
]
Time [d]
Repeating units
01
6912
1416
2023
n = 3n = 4
n = 5n = 6
n = 7n = 8
n = 9
0102030405060708090100
Rel.
peak
inte
nsity
[%]
Time [d]
Repeating units
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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a)
b)
Figure 39 - Fate of individual homologues during aerobic biodegradation (0 - 14 d) of polydispersed PEG 970, measured by (+)-ESI-LC-MS. (a) freshwater media, (b) Artificial seawater media
Although the microorganisms are assumed to be different since we have not defined the microbial population,
the biodegradation of PEG 250 and PEG 970 in seawater is the same as in freshwater with the only difference
the biodegradation in freshwater media being faster. This result coincides with that reported for biodegrada-
tion of alkyl phenol ethoxylates and alkyl benzene sulfonates in seawater [434].
06
914
n = 3n = 5
n = 7n = 9
n = 11
n = 13
n = 15
n = 17
n = 19
n = 21
n = 23
n = 25
0
10
20
30
40
50
60
70
80
90
100
Rel
. pea
k in
tens
ity [%
]
Time [d]Repeating units
0710
14n = 3
n = 5n = 7
n = 9n = 11
n = 13
n = 15
n = 17
n = 19
n = 21
n = 23
n = 25
0
10
20
30
40
50
60
70
80
90
100
Rel
. pea
k in
tens
ity [%
]
Time [d]Repeating units
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When searching for metabolites of PEG 250 and PEG 970, molecules having repeating units of 44 g∙mol-1 were
found. The molecules had an m/z of either 2 less or of 14 more than the m/z of corresponding PEG-Na adducts.
The intensities of these metabolites increased during biodegradation and then decreased again (data not
shown). This suggests the oxidation of one terminal OH-group to the corresponding aldehyde has occurred,
which is the initial step of the biodegradation of PEG 250 and PEG 970 [84;192]. Further oxidation leads to the
carboxylic acid derivative [84;192]. Figure 40 shows the results of the aerobic biodegradation of PEG 2000 in
freshwater media measured by MALDI-TOF-MS.
Figure 40 - MALDI-TOF-MS spectra of polydispersed PEG 2,000 during aerobic biodegradation in freshwater media with WWTP sludge inoculum, matrix was DCTB: (a) sample from day 1, (b) sample from day 6. Numbers indicate repeating units
On day 1 (Figure 40a), each individual PEG-Na-homologue (beside PEG-K-adducts and doubly charged PEG-2Na-
adducts) of the polydispersed PEG 2000 can be seen in the spectrum, covering an m/z range from 1300 to
2600. When the biodegradation is in process on day 6 (Figure 40b), a shift in the chain length and molecular
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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weight of the former PEG 2000 has occurred. Individual PEG homologues cover an m/z range from 600 to 2500,
indicating a loss of long-chain PEGs and a formation of short-chain PEGs. On day 9 (data not shown), PEG could
no longer be detected. Obviously, the biodegradation of PEG 2000 is similar to that of PEG 250 and 970 for the
freshwater media with formation of short-chain homologues.
In artificial seawater, a similar spectrum (Figure 41a) to that for freshwater media (Figure 40a) on day 1 was
obtained for PEG 2000, with the difference that only singly and doubly charged PEG-Na-adducts were detected
due the desalting of the sample and adding of NaTFA as cationization agent.
Figure 41 - MALDI-TOF-MS spectra of polydispersed PEG 2,000 during aerobic biodegradation in artificial seawater media with marine inoculum, matrix was DCTB: (a) sample from day 1, (b) sample from day 14. Numbers indicate repeating units
Although the dissolved organic carbon removal has reached some 50% on day 14 (Figure 36), no short-chain
PEGs were detected at this time (Figure 41b). This is in contrast to the degradation of PEG 2000 in freshwater
where short-chain homologues were found (Figure 40b). Comparing Figure 41b with Figure 41a, a loss of some
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individual PEG homologues in the m/z range 1300 to 1600 can be observed but the long-chain PEGs are still
present. Other samples of PEG 2000 (day 12 and day 16, data not shown) were analyzed, showing the same
distribution of long-chain homologues. On day 20, only PEGs having a molecular weight between 1900 and
2700 g∙mol-1 were present (data not shown), short-chain PEGs could not be detected. Evidently, PEG homo-
logues with molecular weight <1900 g∙mol-1 are degraded preferentially prior to long-chain homologues. The
degradation of PEG 2000 is as fast as that of PEG 250 and PEG 970 in seawater media and was complete after
27 d (Figure 36). A different biodegradation pathway of PEG 2000 in seawater can only explain the difference.
This pathway of PEG homologues having a molecular weight >1900 Da is characterized by a stable molecular
weight pattern during degradation.
MALDI-TOF-MS spectra of PEG 4500, PEG 7400, PEG 10’300 and PEG 14’600 were recorded for freshwater
media. All PEGs mentioned show a shift towards short-chain PEGs when the biodegradation is in progress (data
not shown), meaning that PEGs from molecular weight 250 to 14’600 all have a similar degradation pathway in
freshwater, which is characterized by gradual loss of one oxyethylene group resulting in formation of short-
chain PEGs. During degradation of PEGs 4500 to 10’300 in artificial seawater media, no short-chain PEGs were
formed and detected using MALDI-TOF-MS (data not shown) what suggests a similar degradation pathway as
that of PEG 2000 in seawater. The long-chain PEGs were present until the biodegradation has come to an end,
no change in the distribution pattern was observed. On day 90 of the biodegradation of PEG 10’300, the long-
chain PEGs have disappeared (data not shown), indicating a complete primary degradation of this PEG in artifi-
cial seawater under the simulated conditions.
4.8.1 Adaptation of marine microorganisms
To gain an insight on the biodegradation process two sorts of additional experiments have been performed.
First, the same PEG sample (all PEGs in this study were tested) was spiked at the same concentration (DOC ≡ 20
mg∙L-1) after it was degraded completely by microorganisms in the first (and non-adapted) setup. It could be
observed that all PEGs from 250 to 58’000 g∙mol-1 are degraded right away without a lag-phase and biodegra-
dation is completed within a few days (<6 d) in freshwater media (data not shown). In marine media the same
experiment could not be done due to the lack of complete biodegradation of PEGs >2000 g∙mol-1. Thus it was
investigated if, after a “smaller” PEG molecule was degraded, a PEG with an exceptionally increase in molecular
weight would be degraded more easily than before.
PEG 14’600 g∙mol-1 was spiked after 120 d in a marine test where PEG 4’500 g∙mol-1 was biodegraded first. But
even after 50 additional days no biodegradation of the second PEG could be observed (data not shown). The
same was done with a PEG 10’300 g∙mol-1, which was spiked in a test after 50 days where PEG 250 g∙mol-1 had
been degraded. In this case degradation could be observed (data not shown) but not as quick as after spiking
the same PEG in an adapted medium.
Another experiment with pre-adaptation of marine microorganisms for biodegradation study of poly(ethylene
glycols) with molecular weight distribution above 4000 g∙mol-1 was performed. Therefore 5L of marine water
was taken from filter units from a sea-water aquarium at Luisenpark. The amount of water was squeezed gent-
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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ly out of the filter units. About 10-15 units were needed to recover 5L of water with an enriched population of
microorganisms. The water samples were transported to the Laboratory within one hour after sampling. The
water was filtered using a sieve with a mesh size of 70µm. For dry mass determination 1200mL of the water
sample was centrifuged at 0°C and 12500xg for 20min. The supernatant was discarded and the pellet was
washed twice with artificial sea-water and centrifuged again after each washing step. The pellet was afterwards
transferred to weighing bottles with a previously determined weight. After drying the samples overnight at
110°C the final weight was determined and the dry mass calculated. The sea-water sample was aerated until
further use for about 7 days. The colony forming units were determined with marine agar plates and an appro-
priate dilution. Then the complete water sample was centrifuged in aliquots of about 400mL at 0°C and
12’500xg for 20min. The supernatant was discarded and the pellet was re-suspended in artificial sea-water and
made up to a total volume of 8.5L. The appropriate amount to obtain a final concentration of 20 mg∙L-1 total
carbon of PEG 2000 was added. PEG has a TOC of 540 mg∙g-1 and therefore 37 mg∙L-1 are needed. The sub-
stance dissolves quickly in water. The medium was aerated with carbon dioxide free air and stirred on a mag-
netic stirrer. To prevent too much loss in water by evaporation the bottle was covered with Parafilm® and kept
in the dark at 22±2°C. A sample was taken and dissolved organic carbon was determined to confirm that the
calculated dissolved organic carbon content. Samples were taken in intervals for dissolved organic carbon de-
termination and plating to get colony counts. After the dissolved organic carbon had decreased to less than 4
mg∙L-1 (after 28 days), the complete amount of medium was centrifuged again in aliquots of about 400mL at
0°C and 12’500xg for 20min. The supernatant was discarded and the pellet was re-suspended in freshly pre-
pared synthetic sea-water and made up to 15L. The obtained marine water was used for degradation studies
PEG 7’400 and 10’300 g∙mol-1
As shown in Figure 22 (p.117) the pre-adaptation of microorganisms with PEG 2’000 g∙mol-1 was successful.
With increasing number in colony forming units a decrease of the dissolved organic carbon from the removal of
PEG can be observed. After adaptation and spiking the new medium with PEG 7’400 and 10’300 g∙mol-1, it was
discovered that the adaptation phase does not seem to have any effect on the biodegradation of the PEG 7’400
and 10’300 g∙mol-1 (data not shown).
4.8.2 Experiments with 10-fold increased PEG concentration in marine medium
In order to investigate possible threshold levels in biodegradation properties of the PEGs in marine tests, the
concentration was increased by a factor of 10 and it was observed that the biodegradation rate decreased
drastically by the same factor. The degradation rate during the degradation phase (per day) was calculated for
PEG 2000 at approx. ~5.0% (at 20mg dissolved organic carbon per liter) and ~0.50% (at 200mg dissolved organ-
ic carbon per liter). For PEG 7400 the degradation rate was estimated to around ~2.3% (at 20mg dissolved or-
ganic carbon per liter) and ~0.23% (at 200mg dissolved organic carbon per liter).
When PEG 2000 and PEG 7400 are compared the 3.7 fold increase in molecular weight (from 2000 to 7400)
decreases the biodegradation rate by approx. half for both test concentration in marine medium.
Figure 42 - Marine biodegradation of poly(ethylene glycol) samples at 10-fold increased test concentration (200mg∙L-1
4.8.3 Results from molecular analysis of marine PEG degradation tests
)
After sequencing, the data was analysed automatically. The forward and reverse sequence was compared to
NCBI nBLAST library and the closest hits were than confirmed manually: if the corresponding forward and re-
verse sequences did not give same results, sequence alignment was performed and then again compared to
the database. The results are given in Figure 43.
Figure 43 - Results of sequencing of samples from marine poly(ethylene glycol) biodegradation experiments
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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Green frames in the picture are used to highlight sequences where probability for the identification of the or-
ganism was higher than 98%. Red frames show DGGE bands were organisms were not accurately identified.
Arrows show bands which have been sequenced but no hits were found in NCBI databases. The samples ana-
lysed were taken from biodegradation tests at the end of exposure after 206 days of a special experiment using
10-fold increased concentration of test substance (Figure 42).
It is very interesting to see that biodegradation can in this case not be linked to the data obtained by molecular
analysis. The pattern of isolated strains seems random and it would be anticipated to have similar microorgan-
isms in each of the corresponding test replicates. Also it would be anticipated that some organisms in the blank
controls would be found in test replicates, since each of the replicates and blank controls was prepared from
the same medium. The pattern of the strains shows that probably the microorganisms responsible for biodeg-
radation of the PEGs and also the control substance Na-benzoate have not been detected. It also shows that
marine microorganisms are able to survive for a long time even without nutrients (as is the case in blank con-
trols). This may be due to the carbon storage capacity of marine water and the fact that at least some small
amount of CO2 will always be carried into the vessel and the medium by aeration.
Along with samples from biodegradation experiments marine water and synthetic marine medium were ana-
lysed at begin of exposure to show the conditions prior to the experiment Figure 44. Samples 30-33 were taken
from marine synthetic water prepared as described in materials and methods section and samples 34-36 were
taken from Sylt, Westerland from the surface layer of the North Sea. When these patterns are compared to the
results obtained after the biodegradation test shown above it is very interesting that originally only few strains
can be identified and no corresponding data can be established.
Figure 44 - Results of sequencing of samples from marine water (34-36, left photograph) and marine synthetic medium (30-33, right photograph)
4.9 Biodegradation Tests with Ecoflex
This is the first extensive study of aquatic biodegradability and the first trial of investigating pathways. The
duration of the experiments is extremely long. In marine biodegradation tests this does not seem too much of
a problem, since the microorganisms are used to live and survive in nutrient limited environment but it poses a
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problem for WWTP biodegradation tests. In biodegradation tests the aerobic biodegradation of aliphatic-
aromatic polyester was tested using 5 replicates and synthetic marine medium with inoculum from Luisenpark
Mannheim as well as 3 replicates in WWTP inoculum (freshwater tests).
4.9.1 Aromatic aliphatic polyester biodegradation
The biodegradation in freshwater/WWTP medium was observed for about 500 days. The results are shown in
Figure 45. Even though inoculum suspension was added during this test, no biodegradation could be observed.
The decrease and sharp increase at the end of the test may be possibly due to fluctuations in aeration between
blank controls and test replicates.
Figure 45 - Waste water treatment plant/freshwater biodegradation test results of Ecoflex
In the experiments with freshwater/WWTP inoculum, additional inoculum suspension was added once a month
(5mL, prepared as described before) to ensure continuous availability of microorganism consortia.
The results of the marine biodegradation test are given in Figure 46. In marine tests also inoculum suspension
was added in two blank controls and two test replicates. The blank control assays 3 and 4 as well as test sub-
stance assays 4 and 5 have been spiked with 10 mL of freshly taken sea water from the filter units of the aqua-
rium. The suspension was added on day 475, 538, 594 and 652. Nevertheless it could not be determined that
better or enhanced degradation derived from this procedure when the data of the spiked assays is compared
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
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to the others. The fourth replicate shows falling carbon dioxide evolution after 300-400 days of exposure. This
assay should be excluded from further evaluation; however substance specific analysis may reveal valuable
results.
Figure 46 - Marine biodegradation test results of Ecoflex
Although biodegradation was only observed in two of the six replicates the others show because of their con-
stant progress that the test system basically can operate even for long-time studies. Aliphatic-aromatic types of
polymers have been extensively studied in soil or compost environment before which represents their main
application area. We have observed that marine microorganisms can last very long without additional carbon
sources because of their high surface area to volume ratio and their ability to survive in oligotrophic environ-
ment. These observations led to investigation of long-term biodegradability in order to establish metabolic
pathways of the polymer tested. It was also shown by the data generated from blank controls, control sub-
stance and measurement statistics that these tests can be kept quite stable. The presence of microorganisms
was confirmed as was the presence and change in polymers in the specific test assays using substance specific
analysis [1] and molecular tools. During the process of analyzing these samples the whole test batches were
filtered through 0.45µm filters and analyzed at the end of exposure. The residues from the different test assays
are given in Figure 47. It is very interesting to find much difference in the residues and also in analytical da-
ta [1]. The residues show that there has been lots of algae growth in some of the test vessels even thought the
flasks/bottles were kept mostly in the dark. The high variations found using the different parameters deter-
mined can lead to the conclusion that the differing biodegradation rates may be due to effects in population
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changes. One option would be a predator-prey-dynamic that could result in major changes in biodegradation
rates or even in the effect that no biodegradation is observed or some kind of steady state is reached.
It could be found when GPC data on the extracts was evaluated that in all test assays some breakdown and/or
biodegradation had occurred since a shift in molecular weight distribution was observed when compared to
the Ecoflex extract form synthetic marine medium without inoculum (standard). However, this is not consistent
with the results obtained by biodegradation tests. The influence of loss of mineral medium and different
amounts of test substance can be discarded because all test batches were treated in the same way and same
amounts of samples were taken.
a b c d
e f g h
Figure 47 - Residues of the filtered test assay from the marine Ecoflex long term biodegradation test (a: synthetic test me-dium with Ecoflex powder (without inoculum) as verification of extraction procedures; b: blank control 1; c: test assay 1; d:
test assay 2; e: test assay 3; f: test assay 4; g: test assay 5; h: test assay 6
The amount of medium has been very constant which was confirmed by weighing of the bottles. At the end of
the test when samples for molecular analysis were taken, the amount of medium ranged from 1140-1207 mL in
the test vessels (about 400mL were taken for molecular analysis). It was also confirmed that the correct
amount of polymer was added [1]. GPC showed similar data and also the weighing scoops used to add the
polymer samples were recovered afterwards confirming correct test conditions [1].
4.9.2 Results from molecular analysis of marine Ecoflex degradation tests
Since biodegradation of Ecoflex samples showed quite different kinetics for the replicates in the test, it was
decided to analyze medium samples with molecular tools. The intention was to confirm different microorgan-
ism communities in the replicates as well as in blank controls and control substance (positive control) and
maybe also to show similarities in those replicates where no biodegradation was observed and those which
show some biodegradation at least.
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The results are given in Figure 48. It is very interesting, that no prediction can be made based on this data to
identify possible microorganisms that are able to degrade Ecoflex in marine medium. However it is also inter-
esting, that there can still be found viable microorganisms in blank and positive controls after a very long time.
Some organisms such as Synedra sp. were found in many test assays as well as Haslea sp.
Figure 48 - Results of sequencing of samples from marine Ecoflex biodegradation experiments
When the sequencing data is compared to biodegradation test data, it seems obvious that the organisms that
have been detected cannot be clearly correlated with the biodegradation data. This however unfortunate fact
from this first trial shows that in order to identify biodegrading microorganisms in marine environment more
detailed research might be necessary. On the other hand, industry and contracting parties doing tests for cus-
tomers need much easier and faster methods to be useful an provide valuable data. It is also very interesting
that even in blank and positive controls some microorganisms can be detected but not as many different ones
as were found in the test substance batches. This could mean that there are probable more than one species
that is able to grow on Ecoflex as a substrate even though biodegradation is very low and only notable in two of
the replicates.
4.10 Biodegradation Tests with Ecovio
Two different types of Ecovio were submitted to freshwater (WWTP) and marine biodegradation tests. The
tests were observed for 500 days. Two replicates were set up with Ecoflex again and three replicates were set
up with Ecovio 2099 L BX 8145 and Ecovio 2129-2 L BX 8180, respectively. In freshwater/WWTP tests inoculum
was added once per month using 5mL of the inoculum suspension that is regularly put in OECD 301 A & B tests.
The polymers and the reference substance (Cellulose) were added to give a total organic carbon concentration
of 100 mg∙L-1 in the test. The results of Ecoflex were discarded. No Biodegradation was observed either in
freshwater/WWTP nor in marine water during this 500 day period. The results for Ecovio are given in Figure 49
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for freshwater and in Figure 50 for seawater. Both Ecovio samples are based on Ecoflex and PLA blends. The
part of PLA is higher in Ecovio 2129-2 L BX 8180 than in the other Ecovio type.
Figure 49 - WWTP/freshwater biodegradation test results of Ecovio
For Ecovio 2099 L BX 8145 it can be seen that in both test systems at the end of exposure almost the same
biodegradation degree is obtained even though characteristics are much different. For Ecovio 2129-2 L BX 8180
no biodegradation is observed in both test systems. This is a very interesting observation and assuming that the
Ecoflex part of the blend might be biodegraded it would fit almost the content of Ecoflex in the blends (45 and
19% respectively). Since PLA is known to hydrolyze but not to biodegrade very easily, this would explain why
the biodegradation degree is lower in the second Ecovio type. This could also explain why Ecoflex has not been
easily degraded in the first place. If PLA hydrolyzes and therefore breaks the polymer down to smaller parts,
the Ecoflex part may be much more susceptible to microbial attack than in the pure Ecoflex grade.
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Figure 50 - Marine biodegradation test results of Ecovio
In marine tests it can be observed that the distribution is broader than in the corresponding freshwater test.
This effect can maybe be attributed to the different conditions and the capacity to store carbon dioxide and
buffer the complete system much more than freshwater. Since all these test replicates from both tests were
set up parallel and on the same aeration line, fluctuations in aeration should have the same effect on each
replicate and can be excluded as cause for this observation.
4.10.1 Results from molecular analysis of marine Ecovio degradation tests
Similar to the experiments with Ecoflex, molecular analysis was performed at the end of exposure of the ex-
periments. The results are also similar (Figure 51) and answer not many questions. No clear identification of
possible degraders correlates to the data from biodegradation tests described above. It is interesting that
Rhodococcus sp. where found in all Ecovio replicates and estimated to occur in more or less similar amounts
but in no other test assays or blank or positive controls.
Also a very interesting fact can be seen when blank and positive controls are compared to the data obtained
from samples of marine medium or native se water given in Figure 44. Only few bands are mostly detected for
different blank controls (at the end of exposure) and for the medium and native sea water (at the beginning of
exposure). After the experiment different microorganisms are detected than before and also more precise
bands result from the DGGE experiment. In the test assays this effect seems even more improved.
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Figure 51 - Results of sequencing of samples from marine Ecovio biodegradation experiments
The conclusion could be drawn that generally the density of microorganisms is quite low for each species in the
tested sample and after a certain time some of the organisms have survived and multiplied while others have
not. In the replicates with polymers at least some degradation may occur even though it might be very low and
not detectable with the used test methods but enough for more organisms to survive and for the growth of
colonies. However, in another investigation on PU biodegradation, DGGE showed that communities on PU
surface were less diverse than in soil and only few species found on the PU surface were detectable in the soil.
Also the soil type influences the composition of microorganism communities depending on the pH and organic
carbon. The interesting point is that PU is highly susceptible to soil biodegradation and independently degraded
almost completely in both soil types but by different communities [202]. If this observation is transferred it
could mean that the polymer might help to “enrich” more microorganisms that could not be found before
using the same method of analysis.
4.11 Results on biomass effects on biodegradation tests
4.11.1 Determination of biomass
The results of the different methods for biomass determination are shown in Figure 52. The mean values of
biomass concentration for centrifugation, filtration and Biuret assay are in close correspondence but due to a
possible outlier in the centrifugation data set, the actual median value shows an actual lower distribution.
Comparing the methods, filtration is the more accurate one with a relative standard deviation of about 3%. The
most precise method of all determination techniques is the Biuret assay with a relative standard deviation of
only 2%. The mean and median values are both a bit higher as in the first two methods. The reason for this
effect could be the underestimation of biomass in the dry mass determination when filtration and centrifuga-
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
146|191
tion is applied. The abscission of biomass is less complemented in case of the centrifugation than at the filtra-
tion. Some cells may be retained in the supernatant of the centrifuge tubes. Both methods of biomass deter-
mination are underestimating the biomass concentration as well because of the cells volatile components
could be lost during the process. It appears that the determination of living bacteria using plating techniques is
the most inadequate an underestimating method to ascertain the biomass in municipal activated sludge. This is
due to the fact that nutrient agars are first of all selective. Bacteria with complex nutrient requirements are
may not form colonies. Second , only viable and cultivable (1-15% in WWTP activated sludge [435]) cells will be
detected.
Figure 52 - Statistical comparison of different methods for biomass determination in WWTP activated sludge suspension (box = 25-75 percentile (50% confidence interval); drawn line = median; dashed line = mean value (n=4 replicates per de-
termination method)
The Biuret assay is the most accurate method to determine biomass in municipal activated sludge. But this
method is associated with a high complexity and also costly in terms of time and material. In practice it appears
that the determination of dry mass using filtration is the most suitable method tested for this type of inoculum
suspension because of its fast and accurate method and low-cost applicability. The results of the different de-
termination methods are summarized in Table 39.
Table 39 - Comparison of the results on different methods for biomass determination
Method of determination Centrifugation Filtration Biuret assay Plating technique
mean value 4.25 g∙L 4.23 g∙L-1 4.38 g∙L-1 1.54 g∙L-1
EQUATION 43 - SIGMOID REGRESSION MODEL FOR REFERENCE SUBSTANCE EVALUATION 123
EQUATION 44 - ASSUMED PATH OF BIODEGRADATION OF POLY(ETHYLENE GLYCOLS) 154
BIODEGRADATION OF SYNTHETIC POLYMERS IN THE AQUATIC ENVIRONMENT
174|191
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